DNA methylation machinery as molecular targets for cancer therapeutics

One of the elements commonly seen in cancer is the change in methylation status of the genome. These aberrations in methylation appear to be critical for the neoplastic phenotype and manifest as changes to gene expression of oncogenes and tumour suppressors. In addition to epigenetic alterations, the proteins involved in maintaining the plastic methylation status of the genome, DNA methyltransferases and demethylases, also show methylation-independent protein-protein interactions that have effects on cell cycle progression and proliferation. As changes in gene expression and mitotic regulation are seminal elements of cancer, and because several methylated DNA binding proteins show differential expression in a wide variety of cancers, these proteins serve as prime targets for anticancer therapies. This thesis relates to exploring both current and forthcoming possibilities and mechanisms of utilizing the DNA methylation machinery for pharmacological intervention of cancer. Chapter two deals with an antisense drug, currently in clinical trials, targeted to reduction of DNA methyltransferase 1, the maintenance methylation enzyme in mammalian cells. Our data indicate that the existence of a common truncation mutation of the adenomatous polyposis coli gene seen in some forms of sporadic and familial colorectal cancer may lead to downstream upregulation of DNA methyltransferase 1, as reconstitution of the wildtype protein reduces DNA methyltransferase 1 mRNA and protein. Reduction of the transcripts of this methylation enzyme with an antisense oligonucleotide decreases the tumourigenicity of these colorectal cancer cells, and provides a rationale for use of this drug in colorectal cancer patients and prophylactic treatment of adenomatous polyposis coli mutation-bearing individuals. Chapter three describes the rationale, design, and in vitro and in vivo testing of antisense molecules against the methylated DNA binding protein MBD2. These drugs reduce target mRNA and cancer cell tumourigenicity in vitro and inhibit tumour implantation and result in tumour regression in vivo. Fortuitously, the antisense drugs designed have no apparent toxic effects on normal cell viability and growth, nor on animals subjected to serial dosing. Taken together, these findings provide justification for the continued search for therapeutics targeting the methylation machinery.