Mechanisms of DNA Demethylating Drugs Against Cancer Progression

DNA methylation has been shown over the last several decades to play a critical role in tumorigenesis for almost all cancers. In this chapter, we review the underlying changes in methylation that are found in cancer cells, namely genomic hypomethylation and CpG (or regional) hypermethylation (relative to normal cells). CpG hypermethylation is associated with gene silencing, and a major area of research over the last decade has been the identification of numerous genes that are methylated in sporadic cancers. Many, if not most, tumor suppressor genes that have been identified on the basis of their relationship with familial, inherited cancer syndromes, have been shown to undergo CpG hypermethylation in sporadic, non-inherited cancers. We then discuss the association of CpG hypermethylation with transcriptional silencing and review the mechanism(s) through which methylation regulates transcriptional activity. One under-appreciated (but likely relevant) mechanism of transcriptional control through hypermethylation of promoter elements is the steric hindrance that methyl groups may place upon transcription factor binding. Interestingly, this steric hindrance may occur with transcription factor binding sites (ones that contain the CG dinucleotide), but may also be affected by cytosine methylation adjacent to the putative binding site. One of the critical insights over the last 5–6 years has been the understanding of the inter-relatedness of DNA methylation and chromatin structure, especially as affected by post-translational core histone modifications. This is discussed (along with the potential strategy of combining DNA methylation inhibition with histone deacetylase inhibitors). There are several distinct types of methylation inhibitors including nucleoside inhibitors (such as 5-aza-2′-deoxycytidine, 5-azacytidine or zebularine), non-nucleoside inhibitors (such as procainamide and hydralazine) and antisense oligonucleotides directed against one of the three known DNA methyltransferases (DNMT1, DNMT3a and DNMT3b). Of these three types of DNA methylation inhibitors, most clinical experience has been generated with the nucleoside analogues. These require intracellular phosphorylation and incorporation into the host cell DNA in order to function. The mechanisms of action include trapping of DNA methyltransferases onto the DNA (this is only operant with the nucleoside analogues), re-expression of silenced genes that have growth regulatory or pro-apoptotic effects, and DNA damage (again, probably restricted to nucleoside analogues). Interestingly, there is abundant evidence that methylation alone does not determine which genes are expressed after exposure to methylation inhibitors. While enthusiasm for the clinical strategies investigating the incorporation of these agents into our oncologic armamentarium seems warranted, the final portion of this review expresses an element of caution. There are a number of scenarios (which have an experimental basis) in which DNA hypomethylation may lead to adverse outcomes. First, hypomethylation may lead to enhanced mutation (as is seen in mouse cells engineered to lack one of the DNMT enzymes). Second, DNA hypomethylation could conceivably lead to the re-expression of positive growth regulatory genes (such as matrix metalloproteinases which might enhance the invasiveness and metastatic potential of cancers). Third, a mouse expressing a hypomorphic allele of DNMT1 developed aggressive lymphoid tumors (suggesting that in these cells, DNMT1 may function as a tumor suppressor gene). Fourth, DNA damage (which has been demonstrated with the nucleoside inhibitors of DNA methyltransferases) almost invariably is correlated with enhanced mutagenesis and would conceivably lead to an increased risk of tumorigenesis. Finally, unexpected and unusual toxicities might be expected by the use of these agents such as drug induced lupus seen with procainamide and hydralazine, which is related to DNA hypomethylation in T lymphocytes.