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. 2015 Apr;22(2):131-44.
doi: 10.1530/ERC-14-0454. Epub 2015 Jan 5.

HES5 Silencing Is an Early and Recurrent Change in Prostate Tumourigenesis

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Free PMC article

HES5 Silencing Is an Early and Recurrent Change in Prostate Tumourigenesis

Charles E Massie et al. Endocr Relat Cancer. .
Free PMC article

Abstract

Prostate cancer is the most common cancer in men, resulting in over 10 000 deaths/year in the UK. Sequencing and copy number analysis of primary tumours has revealed heterogeneity within tumours and an absence of recurrent founder mutations, consistent with non-genetic disease initiating events. Using methylation profiling in a series of multi-focal prostate tumours, we identify promoter methylation of the transcription factor HES5 as an early event in prostate tumourigenesis. We confirm that this epigenetic alteration occurs in 86-97% of cases in two independent prostate cancer cohorts (n=49 and n=39 tumour-normal pairs). Treatment of prostate cancer cells with the demethylating agent 5-aza-2'-deoxycytidine increased HES5 expression and downregulated its transcriptional target HES6, consistent with functional silencing of the HES5 gene in prostate cancer. Finally, we identify and test a transcriptional module involving the AR, ERG, HES1 and HES6 and propose a model for the impact of HES5 silencing on tumourigenesis as a starting point for future functional studies.

Keywords: AR; ERG; HES5; HES6; NOTCH; epigenetics; methylation; prostate cancer.

Figures

Figure 1
Figure 1
HES5 promoter methylation is an early event in prostate tumourigenesis. (A) Representation of sections through four cancerous prostates from which multiple tumour cores (T1–T5) and adjacent benign cores (N1) were taken for methylation analysis. Regions in purple indicate histologically malignant foci and different shades of purple indicate tumour foci that appeared unconnected in 3D-sectioning. Sample keys provided are ICGC Prostate UK IDs. (B) Heatmap showing the median tumour over benign methylation changes at regions in the promoter regions of eight candidate genes. (C) Boxplots showing the methylation status at the promoter region of HES5 in the cohort of prostate tumours with multiple tissue cores, adjacent benign and blood DNA samples. Boxplots depict quartiles for probes within promoter region genomic windows, error bars denote 95% CI and data points are shown for values outside 95% CIs. (D and E) Genomic views of DNA methylation in tumour cores compared with adjacent benign tissue for (D) the HES5 gene promoter region and (E) the methylation-positive control GSTP1 gene promoter. Plots show the methylation profiles from multiple tumour foci for Case-006, data are presented as log2 ratio of tumour over benign. Gene promoters and orientation are annotated at the top of each plot.
Figure 2
Figure 2
Validation of HES5 promoter methylation as a common event in two additional independent prostate cancer cohorts. (A) CpG methylation summary of the HES5 promoter as determined by bisulphite sequencing from a representative tumour–normal pair. Each column represents one CpG assayed (n=60), red and blue stacked bars represent the proportion of methylated and unmethylated reads, respectively, at each CpG. Column widths are proportional to sequencing coverage (median=786×). (B) Scatter plot summary of HES5 promoter methylation for 39 tumour–normal pairs. (C) Histogram summary of significance testing for increased HES5 promoter methylation in tumour vs normal sample pairs (n=39 pairs from panel-C; paired Wilcox rank sum test; −log2 P values are plotted to visualise distributions). (D) Boxplot summary of HES5 promoter methylation for 304 tumour and 49 benign prostate samples on Illumina 450k arrays (TCGA data). (E) Histogram summary of significance testing for increased HES5 promoter methylation in TCGA tumour vs normal sample pairs (n=49 pairs from panel-E; paired Wilcox rank sum test; −log2 P values are plotted to visualise distributions). (F) ROC curve for HES5 promoter methylation using data from bisulphite sequencing of 39 tumour normal pairs (A, B and C) and methylation array profiling of 49 tumour normal pairs (D and E).
Figure 3
Figure 3
HES5 expression is repressed by methylation in prostate tumour cells and shows an inverse trend with HES6 expression. (A) Boxplot showing methylation status of the HES5 promoter region in LNCaP prostate cancer cells and PrEC benign prostate cells (triplicates from GSE34340 and singletons from GSE40699). (B and C) Expression of (B) HES5 and (C) HES6 in LNCaP prostate cancer cells treated with the demethylating agent 5-aza-2′-deoxycytidine (Aza-dC) for 24 and 48 h (GSE25346). Expression presented as log2 ratios over control untreated cells. (D and E) Boxplot showing the expression of (D) HES5 and its known target (E) HES6 in a separate cohort of prostatic benign and primary tumour tissue (GSE3325). Boxplots depict quartiles, error bars denote 95% CI and data points are shown for values outside 95% CIs. (F, G, H and I) Scatter plots of gene expression from clinical prostate tumours showing the relationship between (F) HES5 and HES6, (G) HES6 and ERG, (H) HES1 and ERG, (I) HES1 and HES6 (including samples from the cohort shown in Fig. 2b and c). Plots on the left show pairwise relationships between gene expression, dashed quadrant lines indicates the mid-point of expression values for each gene. Plots on the right show the relationship between the level and difference in expression for each pair of genes (using median centred values for each gene). Divergence from the dashed zero line indicates an inverse relationship, red trend lines depict loess regression. (J) Simple models of the putative expression networks in benign prostate, prostate cancer and ERG-positive prostate cancer involving the AR, HES5, HES6, ERG and HES1. Genes are depicted by thick horizontal lines, connecting lines depict transcriptional targets of each encoded transcription factor. Connectors with arrowheads depict positively regulated targets, while connectors with flat ends depict repressed targets. Genes shown in grey depict low/no expression in a given condition. On the HES5 gene open circles depict hypomethylation and filled circles depict hypermethylation. ARGs denotes AR-regulated genes. Model drawn using BioTapestry.
Figure 4
Figure 4
Detailed gene expression time-course analysis, genomic binding profiles and ERG knockdown supports an ARERGHES1HES6 transcriptional cascade. (A, B and C) Gene expression profiles from androgen stimulation and vehicle control time-course experiments using VCaP (ERG-positive) and LNCaP (ERG-negative) prostate cancer cells. Panels on the left show the mean centered transcript profiles (as log2 ratios/average) and panels on the right show bar plots of the expression levels (log2 intensity) for (A) TMPRSS2, (B) HES1 and (C) HES6. Error bars depict CI for each time-point measured. Vertical dashed lines correspond to the ‘change-points’ for gene expression in the VCaP (dark red) and LNCaP (dark blue) time-series. (D and E) Bar plots showing the androgen-induced expression ‘change-points’ for each gene from (D) LNCaP and (E) VCaP androgen treatment time-series (values correspond to the dashed lines in panels A, B and C). (F and G) Genomic binding profiles for ERG, ETV1 and the AR in prostate cells at the (F) HES1 and (G) HES6 gene loci. Genomic binding sites for each transcription factor are depicted by coloured horizontal rectangles. Multiple datasets are included for AR-binding profiles using the labelling scheme ‘factor-sample, study’ (i.e. ‘AR-VCaP, Wei et al. (2010)’ represents the binding profile of the AR in VCaP cells from the study of Wei et al. (2010)). A scale bar is shown at the top together with chromosomal locations and gene locations and orientations are indicated at the bottom of each plot. (H and I) Boxplots showing the expression of (H) ERG and (I) HES1 in VCaP cells under control or ERG knockdown conditions (GSE60771). Significance testing was performed using t-tests, P values annotated on each plot.

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