Background: The chromatin insulator CCCTC-binding factor (CTCF) displays tissue-specific DNA binding sites that regulate transcription and chromatin organization. Despite evidence linking CTCF to the protection of epigenetic states through barrier insulation, the impact of CTCF loss on genome-wide DNA methylation sites in human cancer remains undefined. Results: Here, we demonstrate that prostate and breast cancers within The Cancer Genome Atlas (TCGA) exhibit frequent copy number loss of CTCF and that this loss is associated with increased DNA methylation events that occur preferentially at CTCF binding sites. CTCF sites differ among tumor types and result in tissue-specific methylation patterns with little overlap between breast and prostate cancers. DNA methylation and transcriptome profiling in vitro establish that forced downregulation of CTCF leads to spatially distinct DNA hypermethylation surrounding CTCF binding sites, loss of CTCF binding, and decreased gene expression that is also seen in human tumors. DNA methylation inhibition reverses loss of expression at these CTCF-regulated genes. Conclusion: These findings establish CTCF loss as a major mediator in directing localized DNA hypermethylation events in a tissue-specific fashion and further support its role as a driver of the cancer phenotype.

Loss of imprinting (LOI) is an epigenetic event that relaxes an allele-specific restriction on gene expression. One gene that experiences LOI is the paracrine insulin-like growth factor , which occurs commonly in human prostate tissues during aging and tumorigenesis. However, the relationship between LOI and prostate tumorigenesis has not been established functionally. In this study, we created a mouse model with CTCF-binding site mutations at the imprint control region that abolishes CTCF insulator activity, resulting in biallelic expression that mimics increased levels seen with aging-induced LOI. We found that LOI increased the prevalence and severity of prostatic intraepithelial neoplasia (PIN), a premalignant lesion. Engineering deficiency into our model increased the frequency of PIN lesions in an additive fashion. Prostates harboring LOI displayed increased MAPK signaling and epithelial proliferation. In human prostate tissue arrays, we documented a positive correlation in benign tissues of IGF2 levels with phospho-ERK and phospho-AKT levels. Overall, our results establish that LOI is sufficient on its own to increase rates of neoplastic development in the prostate by upregulating critical cancer-associated signaling pathways..

<p>Supplementary Table S1 details the list of primers and their sequence used in this study.</p>

<p>This contains supplementary figures 1-4. Figure S1 displays H19 mRNA expression quantification and bodyweight measurements of additional phenotypes in the study. The data relates to Figure 1. Figure S2 contains additional IHC measurements for experimental mice for p-ERK and p-AKT. Figure S2 also contains apoptosis quantification data by TUNEL assay. These data relate to Figure 4 of the main text. Figure S2 also conatains mRNA expression measurements of proliferation related genes, related to Figure 5. Figure S3 conatains characterization data of Insulin receptor isoform expression and Igf1-receptor expression in experimental mouse prostates. Figure S4 contains additional data pertaining to human prostate IHC for IGF2, pERK, and pAKT expression, including correlation of these markers in prostate cancer and the average levels in various prostate tissue pathologies. This figure is related to Figure 5 of the main text.</p>

<p>Supplementary Table S1 details the list of primers and their sequence used in this study.</p>