Abstract 4434: CTCF acts as a master regulator to direct epigenetic events important for tumor development and progression
Authors: Damaschke Nathan A. , Yang Bing , Montgomery Anastasia , Svaren John , Roopra Avtar , Luo Jianhua , Keles Sunduz , Jarrard David 
Published: July 2016

Intro

Epigenetic changes, including altered DNA methylation, underlie the development and progression of prostate cancer (PCa). DNA methylation events are frequently seen in PCa, however, how these methylation changes are directed locally and regionally remain an important question. CTCF is a widely expressed chromatin insulator protein which plays important roles in transcriptional regulation, chromatin architecture, and the conservation of epigenetic marks. Previous work has shown that CTCF expression declines in the aging mouse prostate and in human metastatic prostate samples. It is our hypothesis that CTCF loss directs DNA methylation increases at specific loci resulting in gene expression and phenotypic advantages in cancer cells.

Methods

Cell lines with inducible shRNA expression targeting CTCF were created using 9E6/E7 human prostate epithelial cells and PPC1 and LNCaP cancer lines. Knockdowns were confirmed by qPCR and western blotting. DNA methylation was analyzed using MeDIP-chip, utilizing the Affymetrix Cytoscan HD Array and RNA expression analyzed using Affymetrix Human Transcriptome 2.0 Array. Bioinformatic analysis was performed in R 3.2.1 and Bioconductor 3.0. Motif analysis was performed using HOMER. Cell stress included exposure to H2O2 and CoCl2.

Results

We detected 9811 probes with differential methylation after CTCF knockdown (with P&lt;0.01), of which 5,968 probes (60.1%) were hypermethylated. Of the hypermethylated probes, 417 (7%) were near gene transcription start sites and 3,361 (56.3%) were located within transcribed loci. Motif analysis indicated that CTCF target sequences were enriched in regions developing hypermethylation (P&lt;0.01). Pyrosequencing confirmed methylation increases. RNA expression analysis with CTCF knockdown identified 1,394 transcribed loci with significantly altered expression (FDR&lt;0.1). One-third of genes with altered transcriptional levels also contained a differentially methylated probe in its promoter or transcribed region (459/1394; 32.9%). We confirmed numerous genes with promoter hypermethylation corresponding to decreased RNA expression including CASP14, EDN1, EL, FSTL1, and WIPI1. Gene ontology indicates enrichment for anti-apoptotic genes. CTCF knockdown in epithelial and cancer cell lines led to a significantly increased survival of cells faced with hypoxia or oxidative stress.

Conclusion

Reduction of CTCF, an alteration that occurs in the development of advanced PCa, directs the hypermethylation of loci containing CTCF binding sites. These hypermethylated regions correspond to the transcriptional downregulation of genes involved in metabolism, ER stress, and apoptosis. CTCF knockdown results in resistance to hypoxia or oxidative stress. This study demonstrates, for the first time that CTCF acts as a master regulator to direct epigenetic events important for tumor development and progression and is a target for therapy.

Citation Format: Nathan A. Damaschke, Bing Yang, Anastasia Montgomery, John Svaren, Avtar Roopra, Jianhua Luo, Sunduz Keles, David Jarrard. CTCF acts as a master regulator to direct epigenetic events important for tumor development and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4434.