Abstract
Diversification of transcriptomic and epigenomic states may occur during the expansion of colorectal cancers. Certain cancer cells lose their epithelial characters and gain mesenchymal properties, known as epithelial-mesenchymal transition (EMT), and they aggressively migrate into the non-tumorigenic extracellular matrix. In this study, we isolated a subpopulation with accelerated baseline motility (MG cells) and an immotile one (non-MG cells) from a colon cancer cell line (HCT116). Gene expression signatures of the MG cells indicated that this subpopulation was likely an EMT hybrid. The MG cells substantially lost their migratory properties after treatment with a methyltransferase inhibitor, 5-azacytidine, suggesting a role of DNA methylation in this process. Global transcriptome assays of both types of cells with or without 5-azacytidine treatment identified 640 genes, whose expression might be methylation-dependently down-regulated in the MG cells. Global methylation analysis revealed that 35 out of the 640 genes were hyper-methylated in the MG cells. Among them, we focused on the anti-oncogene ZNF350, which encodes a zinc-finger and BRCA1-interacting protein. Notably, ZNF350 knockdown accelerated migration of the non-MG cells, while overexpression of ZNF350 in the MG cells significantly impaired their migration. Finally, pyrosequence analysis together with dual luciferase assays of serially truncated fragments of the ZNF350 promoter (-268 to +49 bp) indicated that three hyper-methylated sites were possibly responsible for the basal promoter activity of ZNF350. Taken together, our results suggest that hyper-methylation of the ZNF350 proximal promoter may be one of the crucial determinants for acquiring increased migratory capabilities in colon cancer cells.
Highlights
Malignant transformation of cancer cells represents the acquisition of specific capabilities including uncontrolled proliferation, resistance to apoptosis, increased migration, and aggressive invasion
We found that 4,178 genes in total were differentially expressed; the MG cells up-regulated 1,800 genes and down-regulated 2,378 genes, compared to the non-MG cells
The mRNA expression changes of these four marker genes were confirmed by Quantitative real-time reverse transcriptionPCR (qPCR) (Figure 2B); the MG cells significantly increased ZEB1 and VIM mRNA levels, while CDH1 and SNAIL mRNA levels were not changed in the cells, compared with those in the non-MG cells
Summary
Malignant transformation of cancer cells represents the acquisition of specific capabilities including uncontrolled proliferation, resistance to apoptosis, increased migration, and aggressive invasion. These phenotypic alterations imply changes in numerous cell signaling pathways and are mainly acquired after alterations of the cell genome. Tumor cells can adapt to changes in microenvironments, resulting in having heterogeneous subpopulations. Certain tumor cells lose their epithelial characters and gain mesenchymal properties, described as epithelial-mesenchymal transition (EMT) [1, 2], and they aggressively migrate into the non-tumorigenic extracellular matrix. Cell migration plays a pivotal role during invasion and metastasis in various types of tumors [1]. Recent studies suggest that epigenetic mechanisms, such as changes in DNA methylation status, are involved in this transition [3]
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