Abstract 2469: A mutated ARID5B protein found in endometrial cancer has a deleterious function with longer half-life

The ARID5B gene encodes a member of ARID transcription factors containing a unique DNA binding domain, ARID, which binds to AT-rich regions. Recently, point mutations of ARID5B, including nonsense, missense, and frame-shift mutations, were found in endometrial cancers. This finding strongly suggests that the ARID5B gene is a novel tumor suppressor candidate gene in this disease. However, detailed functions of wild-type/mutated ARID5B have not been examined. To explore the importance of ARID5B in development of endometrial cancer, the property of ARID5B was characterized. Interestingly, half of the point mutations reported previously were nonsense/ frame-shift mutations, generating premature termination codons (PTCs). Generally, genes with PTCs generate unstable mRNA, which are detected and degraded by the nonsense mediated mRNA decay (NMD) unless the PTCs are located in the last exon or within 50 nucleotide from the last exon-intron boundary. Surprisingly, all the PTCs found in endometrial cancer are located in the last exon or within 50 nucleotide upstream of the last exon-intron of the ARID5B gene, suggesting that these mutated mRNA are stable to generate truncated ARID5B proteins. We also found that the wild-type ARID5B protein has PEST sequences, which make proteins unstable through the ubiquitin-proteasome system. In virtue of the PTCs, the truncated ARID5B proteins lack these PEST sequences, but still have the ARID domain. Our study showed that the truncated ARID5B protein had longer half-life than that of wild-type ARID5B. Furthermore, we showed that the C-terminus of ARID5B had a repressive property, which was confirmed by a reporter gene assay with transient transfection. The C-terminus was deleted in the truncated ARID5B. The data suggest that the wild-type ARID5B could suppress the expression of down-stream target genes, but the truncated ARID5B could not. Taken together, the truncated ARID5B may accumulate in cells due to its longer half-life and inhibit repressive function of the wild-type ARID5B in a dominant-negative fashion. We also performed colony formation assays using an endometrial cancer cell line, Ishikawa. Expression of wild-type ARID5B was strongly suppressed in colonies resistant to G418, comparing with expression of GFP transfected into the cells as a control. In summary, we found that the truncated ARID5B is a long half-life protein without its transcriptional property, which may inhibit the transcriptional property of wild-type ARID5B. Our data suggest that ARID5B plays an important role as a transcriptional repressor in development of endometrial cancer and the mutated ARID5B may inhibit the normal function of wild-type ARID5B in the cancer cells. Citation Format: Norihiko Kawamata, Keiichi Itakura. A mutated ARID5B protein found in endometrial cancer has a deleterious function with longer half-life. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2469. doi:10.1158/1538-7445.AM2014-2469