| DNA methylation, an epigenetic process known to mediate a diverse number of physiological processes such as imprinting, X chromosome inactivation, and transcriptional regulation, can become dysregulated in many human diseases, including cancer. Particularly, in cancer cells, hypermethylation of DNA sequences in the regulatory regions of various genes has been shown to transcriptionally silence those genes, while hypomethylation of DNA sequences has been linked with transcriptional activation, and genomic instability. The central hypothesis explored by this dissertation research is that prostate cancer is characterized by a tremendous dysregulation of DNA methylation processes, leading to both the hypermethylation of certain DNA sequences, as well as the hypomethylation of others, producing the gene expression changes requisite for cancer development and progression. To address this hypothesis, we developed several novel technologies, such as real-time methylation-specific PCR (RT-MSP or MethyLight), high pressure liquid chromatography (HPLC)/tandem mass spectrometry based assays for quantitation of 5-methyl-2'-deoxycytidine (5mC), and high-throughput methyl-binding domain polypeptide based DNA methylation detection assays, and applied them to a large collection of benign and malignant prostate tissues and cell lines in order to determine the nature, extent, and timing of DNA methylation changes during prostate cancer initiation and progression. We found that prostate cancers are characterized by an early wave of hypermethylation changes at the regulatory regions of several genes including GSTP1, APC, RASSF1A, PTGS2, and MDR1, and that these changes are largely maintained during the subsequent progression of prostate cancer to metastatic disease. We showed that, through the use of a novel DNA methylation detection strategy that we term COMPARE-MS, these DNA hypermethylation changes can be exploited for the sensitive and specific detection of prostate cancer. Finally, we demonstrate that prostate cancers also, paradoxically, become undermethylated at several genomic regions, including LINE1 promoter sequences and the regulatory regions of so-called cancer/testis antigens, and eventually begin to show a reduction of overall genomic 5mC content in metastatic prostate cancer lesions. Drawing on these findings, we propose models that may explain the natural progression of prostate cancer from its earliest precurson lesions to life-threatening, disseminated metastatic disease. |
