Transcription factor occupancy limits DNA methylation and determines ICAM1 expression in breast cancer

Abstract

The interaction between TF binding and DNA methylation is increasingly recognized as a key player in the regulation of gene expression. However, the role of this interaction in regulating ICAM1 expression in breast cancer has not been elucidated. CpG methylation in the ICAM1 promoter is negatively correlated with ICAM1 expression, and ICAM1 expression is significantly positively correlated with DNMT and TET3 expression in breast cancer. TF binding attenuates ICAM1 promoter CpG methylation and promotes ICAM1 transcription. DNA methylation regulation enhances ICAM1 expression in breast cancer by promoting the transcription of transcription factors. In terms of mechanisms, RELA and STATs recruit TET3 to prevent DNMT-mediated DNA methylation, thereby maintaining CpG island hypomethylation in the ICAM1 promoter. Therefore, TF occupancy limits DNA methylation and affects ICAM1 expression in breast cancer.

1. Introduction

Intercellular adhesion molecule-1 (ICAM1) is a glycoprotein expressed on the cell membrane that mediates homotypic and heterotypic interactions between individual cells or between cells and the extracellular matrix. It plays crucial roles in various immune functions and in the development, progression, and metastasis of cancer [1–3]. ICAM1 expression is significantly higher in breast cancer patients than in healthy individuals. Additionally, it is more common in patients with triple-negative breast cancer (TNBC) than in patients with other molecular subtypes of breast cancer, such as hormone receptor (HR)-positive and Her2-positive breast cancer [4]. We previously reported that elevated ICAM1 expression in breast cancer cells aggravates cancer bone metastasis by interacting with integrins and activating the EMT pathway in a TGF-β-dependent manner in TNBC tumor-bearing mice [5]. Additionally, tumor-derived exosomal ICAM1 promotes TNBC bone metastasis by inducing CD8+ T-cell exhaustion [6]. Compared with non-TNBC (nTNBC) subtypes, ICAM1 also enhances the lung metastasis of TNBC in tumor-bearing mice through the formation of circulating tumor cell (CTC) clusters [7]. However, the molecular mechanism underlying the differences in ICAM1 expression across subtypes of breast cancer has not been elucidated.

An important epigenetic mechanism, DNA methylation, is a covalent modification of DNA sequences that plays a crucial role in gene expression and genome stability [8]. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), which transfer a methyl group (CH3) from S-adenosyl methionine to the 5-carbon position of cytosine to generate 5-methylcytosine (5mC) [9]. The additional methyl group not only causes water molecules to bulge out around the DNA backbone but also leads to the loss of hydrogen bonds, which loosens the conformation and prevents desired interactions, thereby affecting the normal function of the DNA [10]. The CpG island in the promoter region is usually unmethylated, a state associated with active gene expression, whereas a methylated CpG island in the promoter and/or enhancer region is associated with gene suppression [11–13]. Numerous studies have reported a negative correlation between the degree of DNA methylation in the CpG island in the ICAM1 promoter and the ICAM gene expression level [11–18]. In noncancer diseases such as rheumatic heart valve disease and Graves’ disease, a high level of ICAM1 expression is negatively correlated with the DNA methylation level of its promoter [14–16]. Additionally, low temperatures can induce the hypomethylation of ICAM-1, leading to the overexpression of the ICAM-1 protein and an increased risk of cardiovascular events [17,18]. In patients with cancers such as prostate cancer and breast cancer, a similar negative correlation between the mRNA level of ICAM1 and the DNA methylation level in the ICAM1 promoter has been observed [19–21]. However, the mechanism by which DNA methylation regulates ICAM1 expression in breast cancer remains unclear.

Cell type-specific gene regulation depends on transcription factors (TFs) that directly bind to specific sequences in genomic DNA through their DNA-binding domains [22]. The ICAM1 promoter region contains multiple motifs, suggesting that it can be bound by various TFs that in turn regulate its transcription [23,24]. In fact, some TFs, such as NF-κB, ETS, and STAT, bind to the ICAM1 promoter region and activate its transcription [25,26]. The corresponding