Abstract 1884: Development of a novel VIM-RFP reporter line for colorectal cancer EMT study and drug discovery

Epithelial-to-Mesenchymal Transition (EMT) describes a dynamic and reversible process by which cells lose their epithelial characteristics and acquire mesenchymal properties. Accumulating evidence indicates that EMT displays an array of intermediate states, a phenotype referred to as “partial MET”. EMT is executed in response to signaling pathway molecules and microRNAs (miRNAs) that induce the expression of specific EMT associated transcription factors (EMT-TFs), including Zeb1/2, Snail1/2, and Twist. There is an ever-growing body of research and clinical evidence indicating that EMT plays an important role in cancer cell dissemination and distal metastasis. Therefore, targeting EMT is considered a novel opportunity in anti-cancer treatment and drug development.Vimentin, a hallmark of mesenchymal cells, is of increasing interest as a novel anti-cancer therapeutic drug target. In colorectal cancer (CRC) patients, increased expression of the vimentin protein predicts a poor prognosis. Here, we developed a novel CRC HCT-116 VIM-RFP reporter line using CRISPR/Cas9 technology. In this cell line, the RFP reporter was incorporated into the endogenous vimentin gene just before the stop codon at the last exon, enabling real-time monitoring of EMT states in live cells. The VIM-RFP knock-in allele was confirmed at genomic, transcriptional, and translational levels. Bio-functional evaluation data revealed that miRNA-200 inhibitor treatment induced the increased expression of VIM-RFP, and decreased expression of E-cadherin, a hallmark of epithelial cells. The expression of EMT-TFs ZEB-1 and ZEB-2 was also upregulated upon induction. In addition, we showed that induced VIM-RFP cells displayed increased migration capacity. These data suggested that miRNA-200 inhibitor induced VIM-RFP cells have undergone EMT. Azacitidine, a clinically approved demethylating agent, has been extensively evaluated in a number of clinical trials as a treatment for CRC patients. Studies reported that azacitidine can induce MET in a number of cancer cell lines. We showed that azacitidine treatment of VIM-RFP cells can effectively induce RFP expression, suggesting a potential application of this VIM-RFP reporter line as a platform for drug evaluation and compound screening. Taken together, the HCT-116 Vim-RFP EMT reporter line could be a valuable tool for dissecting the molecular mechanisms underlying EMT and for evaluating or screening compounds targeting EMT in CRC.Citation Format: Weiguo Shu, Diana Douglas, Sangeeta Kumari, Luis Romero, Luis G. Rodriguez, Chaozhong Zou, Robert Newman. Development of a novel VIM-RFP reporter line for colorectal cancer EMT study and drug discovery [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 1884.