Abstract 1045: CRISPR/cas9 mediated generation of an EMT reporter cell line for metastatic breast cancer drug discovery and development

Abstract

Abstract Among women, breast cancer continues to be the most common cancer, with metastasis being the leading cause of mortality in cancer patients around the world (DIZON et al. 2016). Epithelial to mesenchymal transition (EMT) - the process by which epithelial cells shift to the mesenchymal state, has been implicated in many aspects of breast cancer tumorigenesis, metastasis and drug resistance ). The accumulation of a large body of data on the association of EMT with cancer over the years has not resulted in EMT being an active target for therapeutic development. This is due in part to the lack of appropriate in vitro models. Here we have exploited some of the basic biology of EMT, to create an advanced in vitro metastatic breast cancer reporter cell line model for use in both basic research and discovery of new EMT inhibitors. During EMT, E-cadherin protein expression is down regulated in cancer cells in association with the loss of cell-to-cell adhesion, apico-basal polarity and a change to a spindle-shaped morphology. By installing an emerald green fluorescent protein (EmGFP) tag on the C-terminus of the e-cadherin (ECAD) gene in the epithelial BT-474 breast cancer cell line via CRISPR/Cas9 genome editing, end-point or real-time EMT status of cells can be tracked under defined conditions. The EMT reporter cell line was verified at the nucleic acid (genomic and mRNA) and protein levels as well as in cell-based assays. Bio-functional evaluation of the BT-474 ECAD EmGFP cell line, shows that it responds to EMT induction. The subsequent EMT status of cells can be monitored in real time by observing and measuring ECAD EmGFP expression, in addition, the resulting mesenchymal cells have increased invasive capacities. Furthermore, this EMT reporter cell line shows sensitivity to the MEK1/2 inhibitor U0126 - thereby providing the basis for the use of this cell line in high-throughput screening (HTS) applications including the identification of new anti-EMT drugs for metastatic breast cancer. The BT-474 ECAD EmGFP reporter cell line is also a convenient and sensitive model for basic science research on the mechanisms of metastasis. References DIZON, D. S., L. KRILOV, E. COHEN, T. GANGADHAR, P. A. GANZ et al., 2016 Clinical Cancer Advances 2016: Annual Report on Progress Against Cancer From the American Society of Clinical Oncology. J Clin Oncol 34: 987-1011. HAY, E. D., 1995 An overview of epithelio-mesenchymal transformation. Acta Anat (Basel) 154: 8-20. Citation Format: Metewo S. Enuameh, Weiguo Shu, Robert Newman. CRISPR/cas9 mediated generation of an EMT reporter cell line for metastatic breast cancer drug discovery and development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1045.