Abstract 2109: Short isoform ARID5B protein is expressed in endometrial cancer cell lines by usage of the second translation initiation codon

Abstract

Abstract ARID5B, also known as MRF2, is a transcription factor with the ARID-type DNA binding domain. The ARID5B gene generates two types of mRNA isoforms (short and long isoforms) using alternative first exons; the long and short mRNA isoforms encode 130kDa long and 100kDa short isoform ARID5B proteins, respectively. The ARID5B gene is highly mutated in endometrial cancer. Nonsense and frame-shift mutations, generating premature termination codons, are common. This transcription factor may play a role as a tumor suppressor in development of endometrial cancer and the inactivation of the gene by the mutations may contribute to carcinogenesis of this cancer. To characterize functional significance of the gene products in this cancer, we analyzed the ARID5B gene in three endometrial cancer cell lines (Ishikawa, HEC59, and KLE). Two cell lines, Ishikawa and HEC59, predominantly expressed the long isoform mRNA while KLE expressed both the long and short isoform mRNAs. However, surprisingly, all the cell lines predominantly expressed the 100 kDa protein, which was detected by an antibody recognizing the ARID-domain. To characterize the 100kDa protein, we generated rabbit anti-serum recognizing amino-terminus of the long isoform protein, which failed to detect the 100kDa protein. The data suggested that the 100kDa protein lacked the N-terminus of the long isoform ARID5B protein. Internal ribosome entry site (IRES) activity was found in the sequence between the first and second ATGs of the long isoform ARID5B mRNA by the bicistronic reporter gene assay. Our data suggested that the second translation initiation codon was used to generate the short isoform ARID5B protein in the cell lines. To elucidate functional property of this short isoform ARID5B protein expressed in the cell lines, we suppressed the expression of the ARID5B protein by shRNA. The expression was decreased by 50% using the shRNA constructs. Colony formation assay of Ishikawa cells transfected with the shRNA constructs showed that repression of ARID5B short isoform protein decreased the number of the colonies. In summary, the short isoform ARID5B protein generated by usage of the second translation initiation site was indispensable in these endometrial cancer cells. Citation Format: Norihiko Kawamata, Keiichi Itakuara. Short isoform ARID5B protein is expressed in endometrial cancer cell lines by usage of the second translation initiation codon. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2109. doi:10.1158/1538-7445.AM2015-2109