The ARID5B gene is highly mutated in human primary endometrial cancer. Half of the mutations are small insertions/deletions leading to frame-shift mutations in the coding region. It has not been explored how these frame-shift mutations contribute to development of endometrial cancer. In this study, we characterized a frame-shift mutation of the ARID5B gene, commonly identified in endometrial cancer and found that the mutation is associated with aggressiveness of the cancer. We have found that the frame-shift mutations found in human endometrial cancer were clustered in the last exon and within 20 nucleotides from the last exon-intron boundary. Generally, an altered message with a premature termination codon (PTC) generated by a frame-shift mutation is captured and degraded by RNA surveillance system. However, when the PTC is located in the last exon or within 20 nucleotide from the last exon-intron boundary, the altered mRNA with the PTC escapes from the surveillance and produces a truncated protein lacking the carboxy terminus. We hypothesized that the frame-shift mutations of the ARID5B gene produced truncated proteins lacking the C-terminal region of which expression truncated protein lead to unique feature of the cancer. We introduced a small deletion in the last exon of the ARI5B gene of the endometrial cancer cell line, Ishikawa, and established the subline, Ishikawa-1E. The small deletion lead to generation of a PTC. We have confirmed expression of the altered mRNA with the PTC and the truncated ARID5B protein by RT-PCR and western blot analysis, respectively. Although the wild-type ARID5B protein has PEST sequences, which lead to rapid degradation of the protein, the truncated ARID5B lacks the PEST sequences due to the PTC. We hypothesized that the truncated ARID5B is more stable than the wild-type since the former lacks the PEST sequences. Treatment of the parental Ishikawa cells or the subline with cyclohexamide showed that a protein half-life of the truncated ARID5B is longer than the wild-type ARID5B. A wound healing assay showed that the Ishikawa-1E cells migrated faster than the parental cell line, ishikawa. Expression microarray analysis showed that genes associated with metastasis/migration/invasion in cancer were differentially expressed between the parental Ishikawa cells and Ishikawa-1E cells, including GJA1 and CDH12. A xenograft experiment using NSG immunodeficient mice showed that Ishikawa-1E cells grew faster than the parental cells on the mice. In summary, the frame-shift mutations of the ARID5B gene commonly found in human endometrial cancer resulted in production of the truncated ARID5B protein lacking the C-terminus, which lead to aggressiveness of endometrial cancer.
Citation Format: Norihiko Kawamata, Keiichi Itakura. A truncated ARID5B protein generated by a frame-shift mutation leads to aggressiveness of endometrial cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-150.