Genome-Wide Methylome Analysis in Canine Mammary Tumors and Immune Cells Elucidates Epigenetic Tumor Regulation and Its Application to a Malignancy Prediction

Abstract

Canine mammary tumor (CMT) has long been considered as a good animal model for human breast cancer (HBC) due to their pathological and biological similarities. However, only a few aspects of the epigenome have been explored in both HBC and CMT. Moreover, DNA methylation studies have mainly been limited to the promoter regions of genes. Genome-wide dysregulation of CpG methylation accompanies tumor progression and characteristic states of cancer cells, prompting a rationale for biomarker development. Understanding how the archetypic epigenetic modification determines systemic contributions of immune cell types is the key to further clinical benefits. In Chapter 1, the study focuses on genome-wide methylome profiles in canine mammary tumors (CMT) and adjacent normal tissues, particularly highlighting the intron regions as potential targets for epigenetic regulation. The analysis revealed the identification of numerous tumor suppressors and oncogenes. Notably, differentially methylated genes (DMGs), including intron-DMRs (differentially methylated regions), were enriched in cancer-associated biological processes. Interestingly, two PAX motifs, PAX5 (tumor suppressive) and PAX6 (oncogenic), were frequently observed in hyper- and hypo-methylated intron-DMRs, respectively. The study found an inverse correlation between hyper-methylation at PAX5 motifs in the intron regions of CDH5 and LRIG1 genes and their gene expression, while CDH2 and ADAM19 genes with hypomethylated PAX6 motifs in their intron regions showed up-regulation. These findings were validated both in the originally MBD-sequenced specimens and additional clinical samples. Additionally, the study investigated intron methylation and downstream gene expression of these genes in human breast invasive carcinoma datasets from the TCGA database. The regional methylation alterations were conserved in the corresponding intron regions, resulting in altered gene expression in breast cancer. This study provides evidence supporting the conservation of epigenetic regulation in both CMT and human breast cancer (HBC), highlighting the importance of intronic methylation in understanding gene regulation in these diseases. On the other hand, the response of immune cells to cancer plays a crucial role in determining the prognosis of cancer and the efficacy of anticancer treatments. Emerging evidence suggests that immune checkpoints, which are key targets of immunotherapy, are also subject to epigenetic regulation. Consequently, Chapter 2 of this dissertation focuses on investigating the differential DNA methylome profiles in peripheral blood mononuclear cells (PBMCs) obtained from patients with mammary tumors. I conducted methylated CpG-binding domain sequencing (MBD-seq) and investigated the differential methylome landscapes of peripheral blood mononuclear cells (PBMCs) from 76 canines with or without mammary tumors. Through gene set enrichment analysis, it was found that genes involved in the growth and differentiation of T- and B-cells are highly methylated in tumor PBMCs. Furthermore, the study identifies increased methylation and reversed expression in representative marker genes (BACH2, SH2D1A, TXK, UHRF1) that regulate immune cell proliferation. Although there was no dramatic difference in the PBMC methylome between malignant and benign tumors, we devised a machine-learning approach to predict malignancy utilizing our methylome dataset. This study provides valuable insights into the comprehensive epigenetic regulation of circulating immune cells in response to tumors, offering a new framework for identifying benign and malignant cancers through genome-wide methylome analysis. In summary, this dissertation provides a comprehensive exploration of epigenetic landscapes in canine mammary tumors, utilizing genome-wide analysis of methylome and transcriptome profiles both in tumor tissues and peripheral blood mononuclear cells. Cancer regulation through methylation of intronic motifs reveals intriguing similarities between humans and dogs, highlighting the value of companion dogs in advancing our understanding of cancer research. Moreover, the application of immune cell methylome data for predicting malignant tumors presents potential scalability in diagnosing malignancy across various types of cancer in humans as well as dogs. Although additional validation studies are needed for the clinical application of the diagnostic models, I believe these studies will be a crucial cornerstone for treating and diagnosing tumors.