Abstract 476: MicroRNA mediated regulation of ATF5 contributes to homeostasis and benign to malignant transformation in breast cancer cells

The transcription factor ATF5 modulates survival, proliferation, differentiation, and homeostasis. In unstressed conditions, ATF5 has a short half-life and is rapidly degraded due to post-translational modifications. Conversely, ATF5 is upregulated in cells under cellular stress. Furthermore, we have found that ATF5 is elevated and is a survival factor in transformed C6 glioma and MCF7 breast cancer compared to non-transformed cells. Regulation of ATF5 expression is not fully understood. We hypothesized that microRNA (miRNA) play a role in regulating the expression of ATF5 at the 3’ UTR and sought to better understand the role of ATF5 in the transformation to a malignant cell phenotype. To date, no studies have examined the regulation of ATF5 by miRNA. MiRNAs are endogenous small non-coding RNAs 20-25 nucleotides in length that contribute to regulation of gene expression at the translational level. We used in silico modeling programs to identify miRNAs predicted to bind to the 3’ UTR of ATF5. We then aimed to identify the presence of specific miRNA and their ability to downregulate ATF5 during cellular stress and other physiological conditions in vitro. Luciferase reporter assays and immunoprecipitations of ATF5 3’ UTR mRNA and cell lysate were performed and microRNA quantity analyzed via qPCR analysis. Subsequently, transfections of precursor microRNA were carried out in human MCF10A breast epithelial, MCF7 mammary epithelial, and breast adenocarcinoma MDA-MB-231 cell lines, and expression levels of ATF5 were measured under varying physiological conditions via Western Blot analysis. In vitro migration and invasion assays were performed and transformation was studied via MCF10A mammary epithelial cells with inducible SRC. We demonstrate that miRNA are bound to the 3’ UTR of ATF5 and that miRNAs 433-3p and 520b help to regulate the expression of ATF5 under varying stress conditions and at steady state in MCF10A, MCF7, and MDA-MB-231 cells. Additionally, preliminary data indicate that ATF5 is upregulated during transformation of MCF10A cells with inducible SRC and is overexpressed in MDA-MB-231 cells as compared to MCF7 and MCF10A cell lines, and suggest that ATF5 may enhance migration and invasion. Further studies are necessary to elucidate the role of ATF5 in transformation, migration, and invasion. Better understanding of the regulation of ATF5 could have implications in a broad range of human malignancies. Citation Format: Kari Ann Gaither, Bhanupriya Madarampalli, David X. Liu. MicroRNA mediated regulation of ATF5 contributes to homeostasis and benign to malignant transformation in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 476. doi:10.1158/1538-7445.AM2017-476