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

3/30/2019 — 4/3/2019

AACR Annual Meeting 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”.1 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.2 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.3 In colorectal cancer (CRC) patients, increased vimentin protein expression predicts a poor prognosis.4 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.5 Studies reported that azacitidine can induce MET in a number of cancer cell lines.2,6 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.