Abstract 1543: Development of a novel ECAD-EmGFP reporter line for pancreatic cancer MET study and drug discovery

Abstract

Abstract World-wide, metastasis continues to be the leading cause of death in cancer patients1. Although epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) have been implicated in the progression of cancer metastasis and drug resistance, their mechanistic pathway and specific role in disease progression is not fully understood2. As such, the development of a reporter line that enables the real-time monitoring of the changing status of cells will not only aid in dissecting the EMT/MET pathway in the research field, but could also become a robust platform for new cancer drug development. E-cadherin, an adhesion protein expressed in epithelial cells, is upregulated in pancreatic cancer cells during MET and is associated with an increase in tight junctions and apico-basal polarity, as well as a change in morphology3,4. Here, we developed a novel PANC-1 ECAD-EmGFP reporter line using CRISPR/Cas9 genome-editing technology. In this cell line, the emerald green fluorescent protein (EmGFP) reporter was incorporated into the last exon of the endogenous E-cadherin gene, enabling real-time monitoring of MET status in live cells. The ECAD-EmGFP knock-in allele was confirmed at genomic, transcriptional, and translational levels. Functional data revealed that miRNA-200 treatment induced the increased expression of ECAD-EmGFP, and decreased expression of Snail, a transcription factor associated with mesenchymal traits. These changes in marker gene expression, as well as the decrease in invasive capacity upon induction, suggest that cells have undergone MET. This cell line is a valuable tool for dissecting the molecular mechanisms underlying EMT and MET and for evaluating or screening compounds targeting EMT and MET in pancreatic cancer. 1. Dizon DS, et al. Clinical Cancer Advances 2016: Annual Report on Progress Against Cancer from the American Society of Clinical Oncology. J Clin Oncol 34: 987-1011, 2016. 2. Nieto MA, et al. EMT: 2016. Cell 166: 21-45, 2016. 3. Polireddy K, et al, Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation. PLOS ONE 11(10): e0164811, 2016 4. Wong S, et al. E-cadherin: Its dysregulation in carcinogenesis and clinical implications. Critical Reviews in Oncology / Hematology 121: 11- 22, 2018 Citation Format: Diana Douglas, Sangeeta Kumari, Jarkko Huuskonen, Weiguo Shu. Development of a novel ECAD-EmGFP reporter line for pancreatic cancer MET study and drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1543.