Abstract
Mutations in components of the Wnt/β-catenin signaling pathway drive colorectal cancer (CRC) by deregulating expression of downstream target genes including the c-MYC proto-oncogene (MYC). The critical regulatory DNA enhancer elements that control oncogenic MYC expression in CRC have yet to be fully elucidated. In previous reports, we correlated T-cell factor (TCF) and β-catenin binding to the MYC 3′ Wnt responsive DNA element (MYC 3′ WRE) with MYC expression in HCT116 cells. Here we used CRISPR/Cas9 to determine whether this element is a critical driver of MYC. We isolated a clonal population of cells that contained a deletion of a single TCF binding element (TBE) within the MYC 3′ WRE. This deletion reduced TCF/β-catenin binding to this regulatory element and decreased MYC expression. Using RNA-Seq analysis, we found altered expression of genes that regulate metabolic processes, many of which are known MYC target genes. We found that 3′ WRE-Mut cells displayed a reduced proliferative capacity, diminished clonogenic growth, and a decreased potential to form tumors in vivo. These findings indicate that the MYC 3′ WRE is a critical driver of oncogenic MYC expression and suggest that this element may serve as a therapeutic target for CRC.
Highlights
The Wnt/β-catenin signaling pathway controls cellular proliferation and differentiation in the intestinal crypt microenvironment [1]
We first tested the ability of CRISPR/Cas9 complexes expressed from this plasmid to cleave the MYC proto-oncogene (MYC) 31 Wnt responsiveDNA regulatory elements (WREs) in HEK293T cells, which is a cell line that can be efficiently transfected
We demonstrate that a single T-cell factor (TCF) binding element embedded within the MYC 3′ WRE is a critical regulator of MYC expression and that it is required to MYC expression in these cells; what has been lacking in the field, due to difficulties in genetically engineering somatic cell lines, is whether any of these WREs are required for oncogenic
Summary
The Wnt/β-catenin signaling pathway controls cellular proliferation and differentiation in the intestinal crypt microenvironment [1]. Mutations in components of this pathway are highly prevalent in spontaneously arising colorectal cancers (CRCs) and lead to deregulated expression of genes controlled by the β-catenin transcriptional co-activator [1,2]. An understanding of colorectal carcinogenesis requires the identification of Wnt/β-catenin target genes and the WREs that control their expression. Cancers 2016, 8, 52 ribosome biogenesis, and cell cycle progression [6,7]. He et al identified MYC as a direct Wnt/β-catenin target gene in a human CRC cell line and mapped a 51 WRE within the MYC proximal promoter region [8]. Two consensus TCF-binding elements (TBEs) were shown to contribute to 51 WRE activity [8]
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