Genome-Wide DNA Methylation Enhances Stemness in the Mechanical Selection of Tumor-Repopulating Cells.

Wei Huang,Hui Hu,An-Yuan Guo,Qiong Zhang,Ning Wang,Xiangliang Yang

doi:10.3389/fbioe.2020.00088

Abstract

BackgroundDNA methylation plays essential roles in tumor occurrence and stemness maintenance. Tumor-repopulating cells (TRCs) are cancer stem cell (CSC)-like cells with highly tumorigenic and self-renewing abilities, which were selected from tumor cells in soft three-dimensional (3D) fibrin gels.MethodsHere, we presented a genome-wide map of methylated cytosines for time-series samples in TRC selection, in a 3D culture using whole-genome bisulfite sequencing (WGBS).ResultsA comparative analysis revealed that the methylation degrees of many differentially methylated genes (DMGs) were increased by the mechanical environment and changed from 2D rigid to 3D soft. DMGs were significantly enriched in stemness-related terms. In 1-day, TRCs had the highest non-CG methylation rate indicating its strong stemness. We found that genes with continuously increasing or decreasing methylation like CREB5/ADAMTS6/LMX1A may also affect the TRC screening process. Furthermore, results showed that stage-specific/common CSCs markers were biased toward changing their methylation in non-CG (CHG and CHH, where H corresponds to A, T, or C) methylation and enriched in gene body region.ConclusionsWGBS provides DNA methylome in TRC screening. It was confirmed that non-CG DNA methylation plays an important role in TRC selection, which indicates that it is more sensitive to mechanical microenvironments and affects TRCs by regulating the expression of stemness genes in tumor cells.

Highlights

Cancer stem cells (CSCs) are high tumorigenicity (Najafi et al, 2019) and highly self-renewing (Hanahan and Weinberg, 2011) cells that play critical roles in tumor initiation and metastasis (Visvader and Lindeman, 2012)
We aim to study the whole-genome DNA methylation changes during the tumor-repopulating cells (TRCs) selection process in soft matrix to learn more about the underlying epigenetic processes of the potential origin of CSCs (Nimmakayala et al, 2018)
It has been reported that non-CG methylation levels are higher in human ES cells (Thomson et al, 1998) and comprising almost 25% of all methylated cytosine (mC) in H1 stem cells; but, it is absent in fibroblast cell lines (Lister et al, 2009), which implies TRCs at 1-day with strong stemness

Summary

Introduction

Cancer stem cells (CSCs) are high tumorigenicity (Najafi et al, 2019) and highly self-renewing (Hanahan and Weinberg, 2011) cells that play critical roles in tumor initiation and metastasis (Visvader and Lindeman, 2012). Our previous work has developed a mechanical method to select and/or generate CSC-like tumor-repopulating cells (TRCs) from cancer cells by culturing them in soft three-dimensional (3D) fibrin gels (Liu et al, 2012; Tan et al, 2014). Studies have reported that 3D cell culture systems have contributed to tumor invasion/migration/metastasis, angiogenesis, microenvironment, and CSCs (Thiele et al, 2014). Tumor-repopulating cells (TRCs) are cancer stem cell (CSC)-like cells with highly tumorigenic and self-renewing abilities, which were selected from tumor cells in soft three-dimensional (3D) fibrin gels

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