Abstract
BackgroundEpigenetic reprogramming using DNA demethylating drugs is a promising approach for cancer therapy, but its efficacy is highly dependent on the dosing regimen. Low-dose treatment for a prolonged period shows a remarkable therapeutic efficacy, despite its small demethylating effect. Here, we aimed to explore the mechanisms of how such low-dose treatment shows this remarkable efficacy by focusing on epigenetic reprograming at the single-cell level.MethodsExpression profiles in HCT116 cells treated with decitabine (DAC) were analyzed by single-cell RNA-sequencing (scRNA-seq). Functional consequences and DNA demethylation at the single-cell level were analyzed using cloned HCT116 cells after DAC treatment.ResultsscRNA-seq revealed that DAC-treated cells had highly diverse expression profiles at the single-cell level, and tumor-suppressor genes, endogenous retroviruses, and interferon-stimulated genes were upregulated in random fractions of cells. DNA methylation analysis of cloned HCT116 cells revealed that, while only partial reduction of DNA methylation levels was observed in bulk cells, complete demethylation of specific cancer-related genes, such as cell cycle regulation, WNT pathway, p53 pathway, and TGF-β pathway, was observed, depending upon clones. Functionally, a clone with complete demethylation of CDKN2A (p16) had a larger fraction of cells with tetraploid than parental cells, indicating induction of cellular senescence due to normalization of cell cycle regulation.ConclusionsEpigenetic reprogramming of specific cancer-related pathways at the single-cell level is likely to underlie the remarkable efficacy of low-dose DNA demethylating therapy.
Highlights
Epigenetic alterations, represented by aberrant DNA methylation, are frequently observed in various types of cancers [1, 2]
Uniform Manifold Approximation and Projection (UMAP) analysis was conducted using 14,099 genes that can be induced by DAC treatment (UMI counts ≤ 2 in all the 1783 mock-treated cells)
It was Demethylated genes were highly diverse among the DAC-treated clones To examine whether similar or different sets of genes were demethylated in different clones, DNA methylation levels of all the 270,249 genomic blocks in individual clones were compared
Summary
Epigenetic alterations, represented by aberrant DNA methylation, are frequently observed in various types of cancers [1, 2]. Clinical studies used DNA demethylating drugs with the maximum tolerated dose (MTD) aiming at cytotoxic effects, and such studies showed only limited therapeutic efficacy with strong toxicity [12]. The therapeutic effect was first explained by re-activation of a specific tumor-suppressor gene, such as CDKN2A [16], and was shown to be associated with the suppression of tumor-initiating cells by restoration of multiple pathways in tumor cells [17]. Low-dose treatment for a prolonged period shows a remarkable therapeutic efficacy, despite its small demethylating effect. We aimed to explore the mechanisms of how such low-dose treatment shows this remarkable efficacy by focusing on epigenetic reprograming at the single-cell level
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