Abstract 1748: Cyclin D1 harboring T286I mutation promotes oncogenic activation in endometrial cancer.

Abstract

Abstract Introduction. Cyclin D1 is an important regulator of cell cycle progression. Phosphorylation of cyclin D1 at Thr-286 by GSK3beta triggers nuclear export and its cytoplasmic proteolysis via the 26S proteasome. Cyclin D1 overexpression is a common event in various types of human cancers; however,mutations of CCND1 (encoding cyclin D1) have been only reported in endometrial cancer and esophageal cancer. We previously reported T286I mutations of CCND1 in 2 endometrial carcinomas (2.3%). The objective of this study was to identify the functions of T286I mutant of cyclin D1 in endometrial cancer. Materials and methods. T286I mutant cyclin D1 (CD1-T286I) plasmid was generated using wild-type cyclin D1 (CD1-WT) plasmid via site-directed mutagenesis technique. pcDNA3 empty plasmid was used as a control (CD1-Ct). Using these plasmids, the functions of mutant CD1-T286I were analyzed by luciferase assays, immunofluorescence, western blotting and colony formation assays in HEK293T (kidney) and HEC-50B (endometrial cancer) cells, both of which do not possess CCND1 mutations. Statistical analysis was performed by student-T test and p<0.05 was considered statistically significant. Result. Immunofluorescence and western blotting showed predominant accumulation of exogenous CD1-T286I in nucleus, whereas expression of exogenous CD1-WT and endogenous cyclin D1 were observed diffusely in cytoplasm and nucleus. Luciferase assay revealed that pRB1 expression was partially decreased by introduction of CD1-WT and that the pRB1 expression was further decreased by introduction of CD1-T286I (p=0.002). In colony formation assays, introduction of CD1-T286I significantly increased the colony number, compared with CD1-WT (p=0.007) and CD1-Ct. Conclusion. Stabilization and accumulation of mutant cyclin D1 (T286I) in the nucleus might overcome regulation by pRb and induce cell proliferation in certain endometrial cancers. Phosphorylation-dependent nuclear export of cyclin D1 at Thr286 might be critical for prevention of aberrant cell proliferation in human cells. Citation Format: Yuji Ikeda, Katsutoshi Oda, Takahiro Koso, Aki Miyasaka, Tomoko Kashiyama, Osamu Hiraike-Wada, Daichi Maeda, Kei Kawana, Shunsuke Nakagawa, Osamu Tetsu, Yoshihiro Kikuchi, Tomoyuki Fujii, Tetsu Yano, Shiro Kozuma. Cyclin D1 harboring T286I mutation promotes oncogenic activation in endometrial cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1748. doi:10.1158/1538-7445.AM2013-1748