Abstract A45: Connecting alternative transcript usage with differential DNA methylation in aggressive prostate cancer

The information content of the human transcriptome is expanded by the presence of alternative mRNA isoforms produced by alternative transcription initiation, alternative splicing, and alternative polyadenylation. Alternative isoforms can possess distinct oncogenic and tumor suppressive functions by altering gene products and may therefore relate to aggressiveness of disease. A major challenge in prostate cancer biology is the need to distinguish indolent from aggressive disease. Elucidating the molecular determinants of aggressiveness has major implications for developing new genomic diagnostics for clinicians to use, and epigenetic regulation of gene expression that results in more aggressive cancers are compelling drug targets. Here, we hypothesize that differential DNA methylation in gene bodies regulates the production of tumor-promoting alternative transcription events in aggressive prostate cancer. DNA methylation may serve a regulatory role in concert with regulatory proteins and sequence features because transcription start site selection and alternative splicing occur co-transcriptionally. To detect interdependencies between differential DNA methylation and alternative isoforms, we utilized RNA-seq and Illumina HumanMethylation 450K array data produced by The Cancer Genome Atlas for 537 normal prostate and prostate cancer tissue specimens. Reference-guided de novo transcriptome assembly was performed, and fractional usage was computed at both the exon and isoform level for 4,734 expressed genes that contain gene-body differential methylation sites on the 450K array. We identified a set of 120 genes with correlations (|Spearman correlation| > 0.5, FDR adj. p-value 5% between fractional usage when grouped by the top and bottom quintile of DNA methylation) between the exon or isoform usage fraction and gene body CpG site methylation level. Of these, 53 genes contain differential DNA methylation and exon or isoform usage specific to aggressive disease. This gene set includes SEPT9, which is known to have a cell migration promoting alternative promoter regulated by with DNA methylation changes in breast cancer, as well as two genes with prior links to prostate cancer aggressiveness (MT2A and TDRD1). At the exon level, there were 6 cases where at least one differential methylation site associated with first exon usage, 7 with last exons, and 121 with internal exons. At the isoform level, 92 isoforms that associated with gene body DNA methylation contained alternative promoter usage, 80 contained alternative end usage, 71 contained alternative splice donor/acceptor sites, and 82 contained exon skipping/inclusion events. Additionally, 4 isoforms were predicted to be targets for nonsense mediated decay (NMD) by inducing message frameshifts that reveal internal stop codons. The diversity of this distribution suggests that DNA methylation may interact with a variety of different regulatory mechanisms that occur co-transcriptionally. While the 450K array only covers a small fraction of gene body CpG sites, our analysis supports the possibility of alternative isoform usage regulated by prostate cancer-specific and aggressiveness subtype-specific DNA methylation changes. Future work will interrogate the exact nature of the mechanisms by which DNA methylation changes cross talk with regulatory factors in alternative splicing, promoter usage, and polyadenylation. Such mechanisms may be targetable pharmacologically for tumor suppressive effect. Citation Format: Jeffrey M. Bhasin, Angela H. Ting. Connecting alternative transcript usage with differential DNA methylation in aggressive prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A45.