Functional analysis of RIC8A, a candidate tumor suppressor, mutated or inactivated in a subset of breast carcinomas

3651 To identify novel tumor suppressor genes in breast cancer, we integrated data from microarray screening of genomic and transcriptomic outlier genes as well as from non-sense mediated RNA decay analysis of six breast cancer cell lines. The top candidates were analyzed for sequence variation, which resulted in the identification of a truncating mutation of the RIC8A gene in the ZR-75-1 breast cancer cell line. RIC8A (Resistance to Inhibitors of Cholinesterase 8 homolog A) is a guanine nucleotide exchange factor which functions in the G protein signal pathway. It has multiple potential functions in cells commensurate with a possible role in mitosis and cancer. For example, it has been implicated in the asymmetric cell division in Drosophila and C. elegans. It is involved in regulation of microtubule pulling forces during mitotic movement of chromosomes by stimulating G(i)-alpha protein. It also acts as an activator for G(q)-alpha (GNAQ) protein by enhancing the G(q)-coupled receptor-mediated ERK activation. In order to explore its role in cancer, we first determined its expression across our database of microarray data from 250 breast cancers. This indicated that down-regulation of RIC8A was seen in ~7% of primary breast cancers. Interestingly, analysis of the genes coexpressed with RIC8A supported its possible role in mitotic processes in vivo. We then set out to functionally explore the role of hRIC8A in breast cancer cells in biochemical and cellular assays utilizing various fusion proteins of hRIC8A and hRIC8Aβ (an alternative splice form). We show here that hRIC8Aβ interacts with Pin1, the peptidyl-prolyl isomerase implicated in Wnt-signalling, tumorigenecity and cell transformation. We speculate that this association may be one of the ways by which hRIC8A functions in mitosis and may contribute to its association with cancer.