Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene

Abstract

Cancers arise through accumulating genetic and epigenetic alterations, considered relevant for phenotype and approaches to targeting new therapies. We investigated a unique collection of endometrial cancer precursor samples and clinically annotated primary and metastatic lesions for two evolutionary and functionally related transcription factors, CCCTC-binding factor (zinc finger protein) (CTCF) and its paralogue CTCF-like factor, also denoted Brother of the Regulator of Imprinted Sites (CTCFL/BORIS). CTCF, a chromatin modeling- and transcription factor, is normally expressed in a ubiquitous fashion, while CTCFL/BORIS is restricted to the testis. In cancer, CTCF is thought to be a tumor suppressor, while CTCFL/BORIS has been suggested as an oncogene. CTCF mutations were identified in 13%, with CTCF hotspot frameshift mutations at p.T204, all observed solely in the endometrioid subtype, but with no association with outcome. Interestingly, CTCFL/BORIS was amongst the top ranked genes differentially expressed between endometrioid and non-endometrioid tumors, and increasing mRNA level of CTCFL/BORIS was highly significantly associated with poor survival. As aberrant CTCFL/BORIS expression might relate to loss of methylation, we explored methylation status in clinical samples from complex atypical hyperplasia, through primary tumors to metastatic lesions, demonstrating a pattern of DNA methylation loss during disease development and progression in line with the increase in CTCFL/BORIS mRNA expression observed. Thus, CTCF and CTCFL/BORIS are found to diverge in the different subtypes of endometrial cancer, with CTCFL/BORIS activation through demethylation from precursors to metastatic lesions. We thus propose, CTCFL/BORIS as an Epi-driver gene in endometrial cancer, suggesting a potential for future vaccine development.

Figure 1. CTCF mutations, p.T204 hotspot site and association with disease-specific survival in primary endometrial carcinomas

(A) Schematic overview of the CTCF protein showing position of mutations relative to Zink-finger domains (numbered 1-11). Grey diamonds indicate nonsense/frameshift mutation, open diamonds indicate silent mutation, one diamond per mutation. Exons corresponding to translated protein regions are shown below protein structure. CTCF gene; NCBI RefSeq NM_006565, chromosome position chr16:67596310-67673088 on GRCh37/hg19 assembly. (B) Chromatogram of the hotspot mutations (arrows) at position c.610, causing frameshift mutations at corresponding protein position p.T204. Consensus DNA nucleotide sequence along with protein sequence is shown below. (C) Disease-specific survival plot for patients with or without CTCF mutations indicates no significant differences in CTCF mutated vs. non-mutated group.

Figure 2. Increased CTCFL/BORIS mRNA expression level associates with cancer progression and poor survival

(A) CTCFL/BORIS mRNA expression increases significantly through the stages of cancer development and progression from complex atypical hyperplasia (CAH) to primary tumors (PT) to metastases (M). (B) High CTCFL/BORIS mRNA expression level (above upper quartile) identifies endometrial cancer patients with poor disease-specific survival. (C) Bar graph presentation showing proportions of cases with high CTCFL/BORIS expression (above upper quartile) for the stages of cancer development and progression from CAH to PT to M. (D) Box plot presentation of CTCFL/BORIS mRNA levels in relation to systemic disease, i.e. development of recurrence or presence of non-resectable metastatic disease at primary surgery. (E, F) Box plot presentation of CTCFL/BORIS mRNA expression in relation to development of recurrent disease stratified for expression levels in primary tumors; compare high expression (E) to low (F) expression. A significant association between CTCFL/BORIS mRNA level and recurrent disease is seen only for the patient group with initial high CTCFL/BORIS mRNA level in primary lesions (E).

Figure 3. DNA methylation index (MI) shows decreasing level of CTCFL/BORIS promoter methylation from premalignant through primary to metastatic endometrial carcinoma lesions

(A) Overview of CpG-sites marked as lollipops numbered 1-28, on CTCFL/BORIS promoter targeted in the bisulphite sequencing assay. Site marked X not included due to integration in PCR primer. The target region contains two dominant transcription start sites: TSS B and TSS C and exon 1 of the gene (extended overview in Figure S2). (B) Representative bisulphite sequencing samples of normal control (NC), complex atypical hyperplasias (CAH), primary tumors (PT) and metastasis (M). The CTCFL/BORIS promoter shows a gradual loss of methylation through cancer developmental stages compared to normal control (from blood buffy coat). Each horizontal line represents an epiallele, with each individual CpG-site shown as either methylated CpG sites (black circles) or unmethylated CpG site (open circles), with positions corresponding to numbering in A. The vertical stack of epialleles describes analysis of multiple colonies/clones. The controls showed a high degree of methylation of mean value of 95.1 %, while an in vitro PCR generated unmethylated control was 1.8 % methylated, indicating robust analysis. Analysis close to mean values in C) was selected for display. (C) Comprehensive box-plot of MI-values significantly declining through cancer progression with mean values for normal controls of 95.1 %, hyperplasias with atypia 91.2 %, primary tumors 86.2 % and metastases 83.8 %.