Genome-wide DNA methylation and RNA-seq analyses identify genes and pathways associated with doxorubicin resistance in a canine diffuse large B-cell lymphoma cell line

PLOS ONE RESEARCH ARTICLE Genome-wide DNA methylation and RNA-seq analyses identify genes and pathways associated with doxorubicin resistance in a canine diffuse large B-cell lymphoma cell line Chia-Hsin Hsu1¤, Hirotaka Tomiyasu2, Chi-Hsun Liao ID1, Chen-Si Lin ID1* 1 Department of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan, 2 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤ Current address: Department of Biomedical Sciences, Cornell University, Ithaca, NY, United States of America * Abstract OPEN ACCESS Citation: Hsu C-H, Tomiyasu H, Liao C-H, Lin C-S (2021) Genome-wide DNA methylation and RNAseq analyses identify genes and pathways associated with doxorubicin resistance in a canine diffuse large B-cell lymphoma cell line. PLoS ONE 16(5): e0250013. https://doi.org/10.1371/journal. pone.0250013 Editor: Vasu Punj, University of Southern California, UNITED STATES Received: October 2, 2020 Accepted: March 29, 2021 Published: May 7, 2021 Copyright: © 2021 Hsu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Doxorubicin resistance is a major challenge in the successful treatment of canine diffuse large B-cell lymphoma (cDLBCL). In the present study, MethylCap-seq and RNA-seq were performed to characterize the genome-wide DNA methylation and differential gene expression patterns respectively in CLBL-1 8.0, a doxorubicin-resistant cDLBCL cell line, and in CLBL-1 as control, to investigate the underlying mechanisms of doxorubicin resistance in cDLBCL. A total of 20289 hypermethylated differentially methylated regions (DMRs) were detected. Among these, 1339 hypermethylated DMRs were in promoter regions, of which 24 genes showed an inverse correlation between methylation and gene expression. These 24 genes were involved in cell migration, according to gene ontology (GO) analysis. Also, 12855 hypermethylated DMRs were in gene-body regions. Among these, 353 genes showed a positive correlation between methylation and gene expression. Functional analysis of these 353 genes highlighted that TGF-β and lysosome-mediated signal pathways are significantly associated with the drug resistance of CLBL-1. The tumorigenic role of TGF-β signaling pathway in CLBL-1 8.0 was further validated by treating the cells with a TGF-β inhibitor(s) to show the increased chemo-sensitivity and intracellular doxorubicin accumulation, as well as decreased p-glycoprotein expression. In summary, the present study performed an integrative analysis of DNA methylation and gene expression in CLBL-1 8.0 and CLBL-1. The candidate genes and pathways identified in this study hold potential promise for overcoming doxorubicin resistance in cDLBCL. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was supported by the Ministry of Science and Technology of Taiwan (MOST 1072313-B-002-044). Introduction Competing interests: The authors have declared that no competing interests exist. Canine diffuse large B-cell lymphoma (cDLBCL) is one of the most frequently diagnosed malignancies in dogs. It resembles human lymphoma in many essential ways, such as PLOS ONE | https://doi.org/10.1371/journal.pone.0250013 May 7, 2021 1 / 15 PLOS ONE Doxorubicin resistance in canine B cell lymphoma characteristic translocations and molecular abnormalities, as well as similar therapeutic responses to chemotherapy [1]. Multidrug chemotherapy that is doxorubicin-based remains the treatment of choice and extends the life span of dogs affected by cDLBCL; however, cDLBCL remains incurable, with variable survival time and high relapse rates due to drug resistance [1, 2]. Since drug resistance is the major limiting factor in the successful treatment of cDLBCL with doxorubicin-based chemotherapy, understanding the mechanisms causing drug resistance will significantly improve therapeutic strategies for fighting cDLBCL and hence increase the survival rates. Multiple mechanisms contribute to drug resistance, including increased drug efflux, changes in drug metabolism or drug target, and activation of downstream or parallel signal transduction pathways [3, 4]. Increasing evidence has shown that genetic mutations play critical roles in drug resistance; however, the relatively rapid appearance or the reversibility of non-responsiveness to drug treatment cannot be explained by genetics [4]. Also, research has revealed that aberrant DNA methylation may occur at a much higher frequency than gene mutation and therefore have a greater impact on the acquisition of resistance to anticancer drugs [5]. Thus, it is critical to understand the DNA methylation profile of resistant cDLBCL to achieve a breakthrough in cancer treatment. Aberrant DNA methylation has gained increasing attention in the field of cancer. Promoter DNA hypermethylation, which induces gene silencing of tumor suppressor genes, has been generally recognized as one of the critical features of cancer [6]. On the other hand, recent studies have shown that gene body methylation is positively correlated with gene expression [7–9]. Since methylation in both promoter and gene-body regions regulates gene expression, the details of the genome-wide methylation profile of doxorubicin-resistant cDLBCL should be further investigated. The strategy we used here to study the methylation profile is called MethylCap, which is based on the capture of methylated DNA with the MBD domain of methyl-CpG binding protein 2 (MeCP2) [10]. With this strategy, many novel regions that are hypermethylated in biological samples can be revealed [11]. To date, no reports on genome-wide DNA methylation and transcriptome analysis of resistant cDLBCL have been published. CLBL-1, a cDLBCL cell line, has been established from clinical specimens as a powerful research tool for comparative tumor biology and cancer drug development [12]. We previously developed a doxorubicin-resistant CLBL-1, named CLBL-1 8.0, which allowed us to study the doxorubicin resistance of cDLBCL [13]. In the present study, with CLBL-1 8.0 and CLBL-1 as control, we intend to investigate the role of epigenetically dysregulated genes and pathways with a potential role in the drug resistance of cDLBCL. Materials and methods Cell lines CLBL-1 was kindly provided by Dr. Barbara C. Rütgen’s laboratory (University of Veterinary Medicine Vienna, Vienna, Australia). The cells were grown in RPMI medium at 37˚C with 10% fetal bovine serum (FBS; Caisson, USA) in a humidified atmosphere containing 5% CO2. CLBL-1 8.0, a doxorubicin-resistant CLBL-1 established in our previous study (Chen et al., 2019 (...truncated)