Low-dose DNA demethylating therapy induces reprogramming of diverse cancer-related pathways at the single-cell level

Abstract

Background Epigenetic 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. Methods Expression 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. Results scRNA-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. Conclusions Epigenetic reprogramming of specific cancer-related pathways at the single-cell level is likely to underlie the remarkable efficacy of low-dose DNA demethylating therapy.

Single‐cell sequencing in translational cancer research and challenges to meet clinical diagnostic needs

“…First trials with DMTIs yielded promising treatment results, but they also evoked severe side effects 104,105 . Lower treatment dosages of DMTIs were similarly successful, but no major demethylation effect was observed in bulk sequencing experiments in contrast to higher treatment concentrations 106,107 . Analysis of monoclonal populations of the human colon carcinoma cell line HCT116 showed that every clone has a distinct partial demethylation pattern and that the resulting changes in epigenetic regulation are sufficient to slow cancer cell proliferation 107 .…”

“…104,105 Lower treatment dosages of DMTIs were similarly successful, but no major demethylation effect was observed in bulk sequencing experiments in contrast to higher treatment concentrations. 106,107 Analysis of monoclonal populations of the human colon carcinoma cell line HCT116 showed that every clone has a distinct partial demethylation pattern and that the resulting changes in epigenetic regulation are sufficient to slow cancer cell proliferation. 107 This monoclonal analysis exemplified the necessity for single-cell resolution in cancer epigenetics in order to unravel cellular heterogeneity, to device novel therapies and to monitor treatment.…”

“…106,107 Analysis of monoclonal populations of the human colon carcinoma cell line HCT116 showed that every clone has a distinct partial demethylation pattern and that the resulting changes in epigenetic regulation are sufficient to slow cancer cell proliferation. 107 This monoclonal analysis exemplified the necessity for single-cell resolution in cancer epigenetics in order to unravel cellular heterogeneity, to device novel therapies and to monitor treatment. Today, several single-cell methods for DNA methylation and chromatin accessibility are available to study cancer (scATAC-seq, sciATAC-seq, scRRBS-seq, scChip-seq, scTrio-seq).…”

5-Aza-2′-Deoxycytidine (5-Aza-dC, Decitabine) Inhibits Collagen Type I and III Expression in TGF-β1-Treated Equine Endometrial Fibroblasts

“…Demethylating agents are currently used to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). They are also being experimentally used in solid tumours in low dose administration, with very promising results [ 39 , 40 ]. Recent studies have shown an epigenetic involvement in several human fibrotic disorders [ 41 , 42 , 43 , 44 , 45 ].…”

A study on DNA methylation modifying natural compounds identified EGCG for induction of IFI16 gene expression related to the innate immune response in cancer cells

“…Another study showed that histone deacetylase inhibitor trichostatin A and CGK1026 induce IFI16 gene expression in prostate cancer cells ( 33 ). In addition, at a single-cell level, low dose treatment with DNA demethylating agents induces IFI16 gene expression ( 34 ). On the other hand, EGCG is a promoter demethylation-mediated epigenetic drug that induces expression of tumor suppressor genes in colon cancer cells ( 35 ).…”