We describe here an integrated genomic approach for the genome-wide-screening for putative tumour suppressor genes in cancer. Given the multitude of potential target genes in frequently deleted regions of the cancer genome, strategies to narrow down the candidates for detailed study would lead to more efficient identification of cancer genes. Here, we describe how a data integration approach combining three microarray technologies (aCGH, gene expression arrays and NMD microarrays) made it possible to narrow the search for potential mutated genes to three tumour suppressor candidates, one of which, RIC8A, was confirmed to show a truncating mutation, deletion and loss of expression in a breast cancer cell line. Furthermore, while the clinical breast cancers had no clear pathogenetic mutations, a subset of clinical breast cancers associated with aggressive features showed low levels of RIC8A expression indicating that inactivation of this gene may play a role in the pathogenesis of a subset of breast cancers.
This represents the second example of NMD-based discovery identifying a novel truncating mutation in cancer. Our previous studies of prostate cancer cell lines identified EPHB2 as a target for similar deletion-truncation events in the DU-145 cells . Therefore, it is clear that some, but not all mutations are highlighted by the NMD-based strategy, particularly in combination with the aCGH data. Treatment with translation inhibitors, such as emetine, extends the half-lives of numerous transcripts . In addition, nonsense-mediated mRNA decay (NMD) is known to regulate a significant number of genes under physiological conditions , as well as to prevent incorrectly spliced genes from being translated . To avoid selection of stress-response genes or those normally regulated by NMD, we applied stringent filtering criteria to the NMD microarray data from six breast cancer cell lines. These included first, presence of the candidate gene in a deleted region of the genome in that cell line, corresponding with the two-hit Knudson hypothesis of tumour suppressor gene inactivation, and second, a low relative baseline mRNA expression. Using this integrated genomic approach we limited the list of candidates for sequencing to 51. The three top candidate genes with the clearest increase in normalized NMD ratios were sequenced in all cell lines. Of these, RIC8A showed a truncating mutation.
After discovery of a mutation in a cancer cell line, the more challenging task is to ascertain the significance and prevalence of such mutations in clinical samples. The key question here is as to whether the mutation is critical to the pathogenesis of cancer and common across a set of cancers, or possibly a "passenger mutation" selected along with a more important other mutation. Although mutations in the coding region of RIC8A were not found in the cohort of 127 early-stage breast cancers, we cannot rule out that mutations might exist at a low frequency in more advanced cases. Exhaustive pyrosequencing of the entire RIC8A promoter region in early invasive breast cancers as well as in pre-invasive tumours (DCIS) and locally advanced (T3/T4) tumours excluded also promoter hypermethylation as a mechanism for RIC8A gene silencing. Recent estimates of the prevalence of somatic mutations in human cancers are highly variable both within and between classes of cancer . The emerging pattern is that there are relatively few frequently mutated genes, (most of these already known) and a very large number of rare mutations, often highly varying from one case to another . Based on the lack of mutations identified in clinical breast cancer samples as well as the lack of evidence from cancer genome sequencing studies indicating involvement of RIC8A [22, 23], we postulated that RIC8A inactivation could either be a rare genetic event in breast cancer, or possibly, its inactivation in clinical samples could take place more often through other mechanisms, not involving direct truncating mutations or methylation.
RIC8A was originally found by genetic studies in C. elegans and reported to act in vitro as a guanine nucleotide exchange factor (GEF) for G protein alpha subunits . Several studies have since reported that RIC8A is required for G-protein signalling and is involved in centrosome movements during early embryogenesis in C. elegans [24, 25]. In addition, RIC8A was discovered to be required for proper asymmetric division of one-cell-stage in both C. elegans embryos  and Drosophila . However, the physiological role of the mammalian homolog RIC8A in G protein-coupled receptor signalling in intact cells is largely unknown. Yet, a human mitotic phosphorylation motif associated with protein localization to the mitotic apparatus was recently identified in RIC8A, suggesting a role for RIC8A in mitosis . This point to the possibility that RIC8A would play a role in cancer relevant pathways and that its inactivation could be important to the pathogenesis of the disease.
Bioinformatics analysis of microarray data from 251 consecutive breast cancers  showed that low RIC8A expression was significantly associated with hormone receptor negativity (ER and PR) as well as presence of TP53 mutations. A similar analysis of gene expression in 115 tumours from another cohort focussing on early breast cancer indicated a similar, but statistically weaker trend (Figure 3). The association between low RIC8A expression and TP53 mutations was validated by the analysis using qRT-PCR of 38 tumours from the early stage cohort . Furthermore, a previously published analysis of 171 primary breast tumours showed that the chromosome 11p telomeric region is lost at an approximate frequency of 15% . The frequency is highest towards the distal part of the chromosome, where RIC8A is located, and declines towards the centromere. In the same tumours, genes in this region, including RIC8A, were shown to be statistically significantly deleted and underexpressed when combining copy number and gene expression data. Also, this deletion was statistically significantly associated to both recurrence and distant metastases in this patient cohort. There was no significant association with any other clinical parameter. Yet another study showed that 11p15.5-p15.4 copy number loss in primary breast cancers was associated with a higher incidence of recurrent disease in tamoxifen-treated patients . While RIC8A is located in one of the most frequently deleted regions in breast cancer , the deletion obviously involves a large number of genes. We therefore evaluated whether the flanking genes from RIC8A would show similar statistical associations between low expression and ER/PR negativity and TP53 mutation, respectively. For example, this region harbours the SIRT3 gene, which has been associated with cancer [28, 29] and whose expression showed correlation with copy number loss together with RIC8A . However, in our analyses RIC8A showed the strongest association with aggressiveness. Together with the presence of the truncating mutation, this suggests a specific importance of the RIC8A gene in a subset of breast cancers.