MP52-03 DISRUPTION OF CHD8-CTCF CHROMATIN COMPLEX IN PROSTATE CANCER ALTERS DNA METHYLATION PATTERNS

Abstract

INTRODUCTION AND OBJECTIVES: Epigenetic features drive cancer progression, but little is known regarding the targeting of DNA methylation. CCCTC-binding factor (CTCF), Chromodomain helicase DNA-binding protein 8 (CHD8), and Brother of the regulator of imprinted sites (BORIS) are interrelated chromatin proteins involved in the regulation of epigenetic marks. BORIS and CTCF are paralogues which compete for CTCF binding sites, exhibit opposing functions, and require CHD8. We evaluated the role that this complex plays in protecting methylated regions, and investigated their expression in prostate cancer (PCa). METHODS: Doxocycline-inducible CTCF targeting shRNAs were stably introduced into PCa lines PPC1, LNCaP and HPV16 immortalized prostate epithelial cells. Proliferation and cell cycle was assessed using flow cytometry. DNA methylation was tested at CTCF binding sites using quantitative pyrosequencing. To evaluate gene expression in PCa, tissue microarrays consisting of benign (N1⁄496), localized (N1⁄4146), metastatic PCa (N1⁄444) and HGPIN (N1⁄450) were chromogenically stained for CTCF, CHD8, and BORIS. A novel, automated quantitative imaging system VECTRA was used to evaluate epithelial staining in both the nucleus and cytoplasm. RESULTS: Knockdown of CTCF was performed to disrupt this chromatin complex. It had no effect on cell proliferation or apoptosis. However, CTCF loss leads to an increase in methylation across CTCF binding sites within the Cav1 gene and the Igf2-H19 imprinted region. Quantitative protein expression in clinical samples demonstrated a marked downregulation of CHD8 expression in HGPIN (P<0.01), localized (P < 0.001), and metastatic PCa (P < 0.0001) compared to benign. CHD8 expression was downregulated in 52% of primary cancer samples indicating a common alteration. Decreased nuclear CHD8 expression was associated with positive margins (P 1⁄4 0.047), extracapsular extension (P<0.01), and presence of metastases (P1⁄40.025). BORIS expression, as was the BORIS/CTCF ratio, was increased in localized PCa (P1⁄40.03 and 0.03) compared to benign. Heterogeneity of staining was assessed using the Simpson’s coefficient which revealed CTCF expression to be more heterogeneous in cancerous tissue (both P < 0.001), especially with higher Gleason grade (p<0.01). CONCLUSIONS: In the first detailed analysis in cancer, a marked loss of CHD8 expression and increased BORIS/CTCF ratio indicate disruption of this chromatin complex. Loss of this complex results in DNA hypermethylation potentially explaining the targeting of DNA methylation changes in cancer.