Oncogene (2012) 31, 2090–2100 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE An integrated genomic approach identifies ARID1A as a candidate tumor-suppressor gene in breast cancer A Mamo1,9, L Cavallone2,9, S Tuzmen3, C Chabot1, C Ferrario1, S Hassan1, H Edgren4,5, O Kallioniemi4,5, O Aleynikova6, E Przybytkowski1, K Malcolm1, S Mousses3, PN Tonin2,7,8 and M Basik1 1Department of Oncology, Lady Davis Institute, Sir Mortimer B Davis Jewish General Hospital, McGill University, Montreal, Que´bec, Canada; 2Department of Human Genetics, McGill University, Montreal, Que´bec, Canada; 3Pharmaceutical Genomics Division, The Translational Genomics Research Institute, Scottsdale, AZ, USA; 4University of Helsinki, Institute for Molecular Medicine (FIMM), Helsinki, Finland; 5VTT Technical Research Centre of Finland and University of Turku, Medical Biotechnology, Turku, Finland; 6Department of Pathology, Jewish General Hospital, Montreal, Que´bec, Canada; 7Department of Medicine, McGill University, Montreal, Que´bec, Canada and 8The Research Institute of the McGill University Health Centre, Montreal, Que´bec, Canada Tumor-suppressor genes (TSGs) have been classically defined Introduction as genes whose loss of function in tumor cells contributes to the formation and/or maintenance of the tumor phenotype. Cancers occur as a result of dysregulation in the TSGs containing nonsense mutations may not be expressed function of oncogenes and tumor-suppressor genes because of nonsense-mediated RNA decay (NMD). We (TSGs). TSGs have been classically defined as genes combined inhibition of the NMD process, which clears whose loss of function in tumor cells contributes to the transcripts that contain nonsense mutations, with the applica- formation and/or maintenance of the tumor phenotype tion of high-density single-nucleotide polymorphism arrays (Presneau et al., 2003). Various mechanisms of inactiva- analysis to discriminate allelic content in order to identify tion of TSGs have been reported and can include candidate TSGs in five breast cancer cell lines. We identified deletion of one allele through chromosomal non- ARID1A as a target of NMD in the T47D breast cancer cell dysjunction and/or intra-chromosomal rearrangement, line, likely as a consequence of a mutation in exon-9, which and mutational inactivation of the remaining allele introduces a premature stop codon at position Q944. (Presneau et al., 2003). TSGs have been identified in ARID1A encodes a human homolog of yeast SWI1, which cancer both as a result of a search for germline is an integral member of the hSWI/SNF complex, an ATP- mutations in hereditary cancers and the molecular dependent, chromatin-remodeling, multiple-subunit enzyme. characterization of somatic deletions and mutations in Although we did not find any somatic mutations in 11 breast tumor tissue RNA and DNA (Vogelstein and Kinzler, tumors, which show DNA copy-number loss at the 1p36 locus 2004). However, TSGs containing nonsense mutations adjacent to ARID1A, we show that low ARID1A RNA or may not be detectable using RNA-based next-genera- nuclear protein expression is associated with more aggressive tion re-sequencing because RNA containing such breast cancer phenotypes, such as high tumor grade, in two aberrant transcripts is eliminated by the nonsense- independent cohorts of over 200 human breast cancer cases mediated RNA decay process (NMD) (Noensie and each. We also found that low ARID1A nuclear expression Dietz, 2001). Indeed, an interesting strategy to discover becomes more prevalent during the later stages of breast potential TSG candidates was elaborated by Noensie tumor progression. Finally, we found that ARID1A re- and Dietz (2001) and adapted by Huusko et al. (2004). expression in the T47D cell line results in significant inhibition Noensie and Dietz (2001) proposed that it would be of colony formation in soft agar. These results suggest that possible to discover nonsense mutation containing ARID1A may be a candidate TSG in breast cancer. mRNA by inhibiting the NMD process. In this way, Oncogene (2012) 31, 2090–2100; doi:10.1038/onc.2011.386; transcripts containing nonsense mutations are stabilized published online 5 September 2011 and selectively increase in quantity relative to non- mutated transcripts. Huusko et al. (2004) then combined Keywords: ARID1A; nonsense-mediated mRNA decay; NMD inhibition with array comparative genomic tumor-suppressor gene; breast cancer hybridization (aCGH) in order to enrich for the selection of candidates mapping to deleted regions in prostate cancer cell lines, thereby identifying EPHB2 as Correspondence: Dr M Basik, Department of Oncology, Lady Davis a novel candidate TSG in prostate cancer. Institute, Sir Mortimer B Davis Jewish General Hospital, McGill We have adapted the approach of Huusko et al. by University, 3755 Cote Ste-Catherine, Montre´al, Que´bec, Canada integrating the allelic content inferred from high-density H3T 1E2. genotyping single-nucleotide polymorphism (SNP) E-mail: [email protected] arrays to identify candidates TSGs in five breast cancer 9These authors contributed equally to this work as first co-authors. Received 16 February 2011; revised 6 July 2011; accepted 26 July 2011; cell lines. Here we report the discovery of ARID1A as a published online 5 September 2011 candidate TSG in the T47D breast cancer cell line by Inactivation of ARID1A in breast cancer A Mamo et al 2091 using this approach. ARID1A encodes a human were identified. The percentage of total transcripts homolog of yeast SWI1, which contains a DNA-binding mapping to LOH regions with an NMD ratio >2 motif (AT-rich interactive domain, ARID) and is an ranged from 1.4 to 4% of all transcripts (Supplementary integral member of the hSWI/SNF complex, an ATP- Figure 2). Reasoning that under-expression is a pheno- dependent, chromatin-remodeling, multiple-subunit en- type of classical TSGs, we then selected candidates zyme (Takeuchi et al., 1997, 1998; Dallas et al., 1998, showing levels of gene expression below the overall 2000; Wang et al., 2004; Wilsker et al., 2004; Patsialou median value of expression for each cell line. Using this et al., 2005). An increasing body of evidence has strategy, the number of candidates decreased to 0.6– demonstrated that SWI/SNF complexes have important 1.9% of total transcripts depending on the cell line roles in gene regulation (Winston and Carlson, 1992; (Supplementary Figure 2). To prioritize candidates for Carlson and Laurent, 1994; Hirschhorn et al., 1995; further analysis, we selected those genes, which showed Kennison, 1995; Sudarsanam et al., 1999, 2000), cell an NMD ratio >2 uniquely in one of the five breast proliferation (Nagl et al., 2005, 2006, 2007) and cancer cell lines. The rationale was based on the development (Carlson and Laurent, 1994; Kennison, assumption that if one gene harbors a nonsense 1995). Several genes encoding hSWI/SNF components mutation in a given cell line, it would be very unlikely have been associated with tumorigenesis (Medina and that the same gene would have a nonsense mutation in Sanchez-Cespedes, 2008; Roberts and Biegel, 2009; another cell line of a small sample set because of the very Rodriguez-Nieto and Sanchez-Cespedes, 2009). More- low frequency of nonsense mutations in tumor cells over, Huang et al. (2007) reported that an antisense (Sjoblom et al., 2006). Applying this selection strategy to cDNA resulting from a genomic rearrangement invol- all five breast cancer cell lines led to the selection of 53 ving the ARID1A gene in a primary breast carcinoma candidate TSGs, or 0.03% of the total number of could transform NIH3T3 cells. During the course of our transcripts on this microarray platform (Supplementary investigation, mutations in the ARID1A were reported Figure 1 and Table 1). to occur in up to 50% of ovarian clear cell carcinomas Sequencing of the 53 candidate genes was begun in the (Jones et al., 2010; Wiegand et al., 2010). Thus ARID1A respective cell lines used for their selection. We began appears to show all of the hallmarks of a classical validating our list starting with genes that have been TSG (Presneau et al., 2003). reported to possess potential tumor-suppressive function We also report on ARID1A expression in human or to be involved in breast cancer. A total of 15 genes breast cancers. We found that low ARID1A RNA and/ were sequenced before we found a nonsense mutation in or nuclear protein expression is associated with more CDH1 in the MDA-MB-453 cell line. CDH1 encodes aggressive breast cancer phenotypes. Finally, we found cadherin-1 and is known to be mutated in the MDA-MB- that ARID1A re-expression in the T47D breast cancer 453 cell line (Forbes et al., 2010), a mutation, which was cell line results in significant inhibition of colony verified in our laboratory by DNA sequencing. CDH1 or formation in soft agar. These results suggest that the cadherin is a well-known TSG, responsible for a ARID1A may be a TSG in breast cancer, and that it hereditary cancer syndrome (Campeau et al., 2008). warrants further investigation as a potential diagnostic As Huang et al. (2007) reported that the ARID1A and therapeutic marker in breast cancer. gene was genomically rearranged in a primary breast carcinoma, we also focused on the ARID1A gene in our list. We identified a nonsense mutation, c.944C>T, in exon-9 in the ARID1A gene, which introduces a Results premature stop codon at amino-acid position 944 (Figure 1a). We confirmed that the RNA levels of NMD inhibition combined with genomic analysis identifies ARID1A were very low and increased when the T47D ARID1A as a potential candidate TSG in breast cancer cell line was treated with emetine, the NMD inhibitor A modification of a strategy described by Huusko et al. (Figure 1b). ARID1A is located at chromosomal band (2004) was used to identify transcripts with nonsense 1p36.11, mapping within the LOH region shown by the mutations.