Summary A critical need in understanding the biology of prostate cancer is characterizing the molecular differences between indolent and aggressive cases. Because DNA methylation can capture the regulatory state of tumors, we analyzed differential methylation patterns genome-wide among benign prostatic tissue, low grade, and high grade prostate cancer and found extensive, focal hypermethylation regions unique to high grade disease. These hypermethylation regions occurred not only in the promoters of genes, but also in gene bodies, and at intergenic regions that are enriched for DNA-protein binding sites. Integration with existing RNA-seq and survival data revealed regions where DNA methylation correlates with reduced gene expression associated with poor outcome. Regions specific to aggressive disease are proximal to genes with distinct functions from regions shared by indolent and aggressive disease. Our compendium of methylation changes reveals crucial molecular distinctions between indolent and aggressive prostate cancer.
A major challenge in the clinical management of prostate cancer is the inability to definitively diagnose indolent versus aggressive cases. Contributing to this challenge is a lack of basic science understanding of the molecular basis behind aggressiveness subtypes in prostate cancer. DNA methylation is the epigenetic addition of a methyl group to the DNA base cytosine and has been found to regulate cell proliferation and environmental adaptation. We hypothesized that DNA methylation changes are a mechanism by which an aggressive cancer attains phenotypes that distinguish it from indolent cases via disruption of regulatory networks. This hypothesis was tested by comparing DNA methylation between benign prostate and both low grade (Gleason score 6) and high grade (Gleason score 8 to 10) groups. Methylome-wide next generation sequencing was performed on formalin-fixed paraffin embedded (FFPE) samples from radical prostatectomy cases using MBD-isolated genome sequencing (MiGS). Global clustering analyses showed some separation between the three groups, but also indicated some heterogeneity. Specific differentially methylated regions (DMRs) were detected between the three groups. The two most prevalent were hypermethylation specific to high grade (491 DMRs at 4% false discovery rate) and hypermethylation shared between both cancer subtypes (1334 DMRs at 1% false discovery rate). Statistical and computational analysis of this data set was non-trivial and the software developed to perform the analysis has been made available. High grade specific DMRs are abundant in intergenic and gene body contexts, and occur less frequently in promoters and CpG islands than DMRs shared between both subtypes. Intergenic DMRs are enriched for putative functional elements (ChIP-seq peaks and DNaseI hypersensitive sites from the ENCODE project) suggesting that these changes may have functional impacts as local or regional enhancers regulating multiple genes. To analyze this in more detail, DNA-protein binding sites for specific factors were compared between 5 spatial contexts. Differential patterns of enrichment were observed for regulatory binding factors between promoters, intergenic regions, and gene 3' ends. A de novo motif search revealed 8 frequent sequence motifs in intergenic DMRs that are not similar to any known binding motifs, indicating possible binding sites for as yet unknown factors. For DMRs that are proximal to genes, an interaction network based on curated pathway data was built using the GeneMANIA Cytoscape plugin. Gene ontology enrichment was observed for “positive regulation of cell motility” (Q-value=1.4e-07, 22 of 296 genes in term) and “regulatory region DNA binding” (Q-value=6.7e-07, 18 of 255 genes in term) within this network. Interestingly, each GO term appears as a distinct cluster on the interaction network. While the specific regulatory action of these DMRs and exact target genes in aggressive cancer remains to be determined experimentally, this work has established the presence of focal hypermethylation that distinguishes indolent and aggressive prostate cancer. The high frequency of intergenic but functionally enriched genomic contexts and functional enrichment of these DMRs at genic sites for transcription factors suggests that they regulate aggressiveness in a complex manner through secondary and tertiary effects. This study provides a first glimpse at molecular differences between indolent and aggressive prostate and future work may lead to the development of biomarkers and treatments based on this knowledge. Citation Format: Jeffrey M. Bhasin, Lars Matkin, Margaret G. Taylor, Byron H. Lee, Cristina Magi-Galluzi, Eric A. Klein, Bo Hu, Yaomin Xu, Angela H. Ting. DNA methylation sequencing of prostate tumors: Possible molecular mechanisms for cancer aggressiveness. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-24.
5-aza-2’-deoxycytidine (DAC) is approved by the US Food and Drug Administration for the treatment of myelodysplastic syndrome. It is also being tested in Phase I and II clinical trials for efficacy against other malignancies, including brain, prostate, breast, and lung cancers. Identifying modifier genes that could confer resistance to DAC treatments is, therefore, essential in realizing the full potential of DAC as a chemotherapeutic agent. DAC is a cytosine analog that is incorporated into newly synthesized DNA, where it can prevent propagation of DNA methylation by irreversibly trapping DNA methyltransferases. The consequent reversal of epigenetic silencing of tumor suppressor genes contributes to tumor cell death in the therapeutic context. Previous studies have identified relatively few determinants of DAC resistance, including the equilibrative nucleoside transporters, deoxycytidine kinase, and cytosine deaminase. While these genes are logical candidates because of their functions in cellular pyrimidine uptake and metabolism, our large-scale, unbiased random insertional mutagenesis screen in human cancer cell lines have identified additional, unexpected loci involved in DAC resistance. Our initial screen produced 9 mutant clones resistant to DAC at concentrations of 0.1μM and 1μM. In particular, several of the insertional mutants targeted components of the DNA damage response pathway, including ubiquitin conjugating enzymes and Fanconi anemia complementation group genes, indicating that the primary mechanism of killing by DAC may not be DNA demethylation. This is consistent with the observation that DNA double-strand breaks are among the consequences of DAC treatments. Ongoing functional studies of the mutant clones also suggest that cancer cells with increased efficiency at repairing genomic DNA damage may survive DNA demethylation in the presence of DAC. These results not only inform us of the mechanism of action of DAC-mediated cytotoxicity, our findings also have important clinical implications as these genetic modifiers are also valuable biomarkers and therapeutic targets for achieving personalized cancer treatments involving DAC. Citation Format: Angela H. Ting, Lars Matkin. Unconventional genetic determinants of resistance to 5-aza-2’-deoxycytidine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4620. doi:10.1158/1538-7445.AM2013-4620
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