Bladder cancer (BC) is the tenth most frequent cancer worldwide and is associated with high mortality when diagnosed in its most aggressive form, which is not reverted by the current treatment options. The disruption of normal epigenetic mechanisms, namely, DNA methylation, is a known early event in cancer development. Consequently, DNA methyltransferase (DNMT) inhibitors constitute a promising therapeutic target for the treatment of BC.
1. DNA Methylation
DNA methylation is the covalent addition of a methyl group at the 5-position carbon of cytosine in a cytosine–phosphatidyl–guanine (CpG) dinucleotide
[17][1]. Methylation occurs mainly at CpG islands—CpG-rich regions (at least 50% of cytosines and guanines) in the genome with a size larger than 200 bp
[26,27][2][3]. Moreover, methylation can also be found in repetitive sequences such as retrotransposon elements and centromeres, in the X chromosome (leading to its inactivation) and genomic imprinting
[28][4]. Approximately 29,000 CpG islands can be found in the human genome, most commonly in the promoter regions, close to the transcription starting site (TSS) or first exons
[27][3]. Remarkably, 75% of cytosines in CpG dinucleotides dispersed throughout the genome are methylated, whereas cytosines of CpG islands located within gene promoters remain mostly hypomethylated
[27,29][3][5]. Promoter DNA methylation is classically associated with transcription repression by inducing binding of transcriptional repressors or hampering binding of transcriptional factors
[15,17][1][6]. In fact, a family of methyl-CpG-binding proteins (MBPs) intervenes in gene silencing by binding to methylated CpGs and recruiting histone modifier enzymes to establish histone post-translation modifications, which further sustain transcriptional repression
[30][7]. DNA methylation can also be found in CpG island shores, 2-kb areas upstream of a CpG island, displaying lower CpG dinucleotide density. CpG island shore methylation is also associated with transcriptional repression
[31][8]. On the other hand, methylation in the gene body was shown to stimulate transcription elongation and to have an impact on splicing, with exons disclosing higher methylation levels than introns
[28,32][4][9]. Furthermore, tissue-specific methylation seems to be more frequent in intragenic CpG islands
[33][10]. CpG islands in enhancers also influence gene regulation, i.e., hypermethylation is associated with loss of enhancer marks resulting in gene silencing
[34][11].
2. DNA Methylation as a Therapeutic Target in BC
2.1. Preclinical Studies
A summary of the preclinical studies testing DNMT inhibitors in BC is depicted in
Table 1. The effects of 5-aza were evaluated in cell lines in vitro and in a tumor xenograph model. 5-Aza was shown to inhibit cell proliferation and arrest cells at G0/G1, whereas volume and weight of tumor xenografts in mice were reduced. Interestingly, DNMT3a and DNMT3b expressions were also reduced after 5-aza treatment, causing the re-expression of hepaCAM, a TSG
[56][12]. DAC treatment also led to an increase in hepaCAM expression in T24 and BIU87 cells, associated with arrest at G0/G1 phase
[123][13]. In a canine model of invasive urothelial carcinoma, 5-aza disclosed anti-tumor effects, with 22.2% of the dogs demonstrating partial response and 50% depicting stable disease
[124][14].
Table 1.
Summary of pre-clinical studies targeting DNMTs in bladder cancer (BC).