Development of a Novel VIM-RFP Reporter Line for Colorectal Cancer EMT Study and Drug Discovery

Pipette dropping clear media into cells well plate; 24 well plate in background.

AACR Annual Meeting 2019

Atlanta, Georgia, United States

March 30, 2019


Epithelial-to-mesenchymal transition (EMT) describes a dynamic and reversible process where 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 EMT”. 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 clinical evidence and an ever-growing body of research 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 (VIM), a hallmark of mesenchymal cells, is of increasing interest as a novel anti-cancer therapeutic drug target. In colorectal cancer (CRC) patients, increased vimentin protein expression predicts a poor prognosis. Here, we developed a novel CRC HCT-116 VIM-RFP (ATCC CCL-247EMT) reporter line by using CRISPR/Cas9 genome-editing technology. In this cell line, the red fluorescent protein (RFP) reporter was incorporated into the endogenous VIM 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. Functional data revealed that miRNA-200 inhibitor treatment induced the increased expression of VIM-RFP, and decreased expression of E-cadherin (CDH1), 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 HCT-116 VIM-RFP cells displayed increased migration capacity. These data suggested that miRNA-200 inhibitor induced HCT-116 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 HCT-116 VIM-RFP cells can effectively induce vimentin-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 will be a valuable tool for dissecting the molecular mechanisms underlying EMT and for evaluating or screening compounds targeting EMT in CRC.

Download the poster to discover more about development and application of the HCT-116 VIM-RFP EMT reporter line.