ERG Induces Epigenetic Activation of Tudor Domain-Containing Protein 1 (TDRD1) in ERG Rearrangement-Positive Prostate Cancer

Background Overexpression of ERG transcription factor due to genomic ERG- rearrangements defines a separate molecular subtype of prostate tumors. One of the consequences of ERG accumulation is modulation of the cell’s gene expression profile. Tudor domain-containing protein 1 gene (TDRD1) was reported to be differentially expressed between TMPRSS2:ERG-negative and TMPRSS2:ERG-positive prostate cancer. The aim of our study was to provide a mechanistic explanation for the transcriptional activation of TDRD1 in ERG rearrangement-positive prostate tumors. Methodology/Principal Findings Gene expression measurements by real-time quantitative PCR revealed a remarkable co-expression of TDRD1 and ERG (r2 = 0.77) but not ETV1 (r2<0.01) in human prostate cancer in vivo. DNA methylation analysis by MeDIP-Seq and bisulfite sequencing showed that TDRD1 expression is inversely correlated with DNA methylation at the TDRD1 promoter in vitro and in vivo (ρ = −0.57). Accordingly, demethylation of the TDRD1 promoter in TMPRSS2:ERG-negative prostate cancer cells by DNA methyltransferase inhibitors resulted in TDRD1 induction. By manipulation of ERG dosage through gene silencing and forced expression we show that ERG governs loss of DNA methylation at the TDRD1 promoter-associated CpG island, leading to TDRD1 overexpression. Conclusions/Significance We demonstrate that ERG is capable of disrupting a tissue-specific DNA methylation pattern at the TDRD1 promoter. As a result, TDRD1 becomes transcriptionally activated in TMPRSS2:ERG-positive prostate cancer. Given the prevalence of ERG fusions, TDRD1 overexpression is a common alteration in human prostate cancer which may be exploited for diagnostic or therapeutic procedures

TMPRSS2-ERG -specific transcriptional modulation is associated with prostate cancer biomarkers and TGF-β signaling

<p>Abstract</p> <p>Background</p> <p><it>TMPRSS2-ERG </it>gene fusions occur in about 50% of all prostate cancer cases and represent promising markers for molecular subtyping. Although <it>TMPRSS2-ERG </it>fusion seems to be a critical event in prostate cancer, the precise functional role in cancer development and progression is still unclear.</p> <p>Methods</p> <p>We studied large-scale gene expression profiles in 47 prostate tumor tissue samples and in 48 normal prostate tissue samples taken from the non-suspect area of clinical low-risk tumors using Affymetrix GeneChip Exon 1.0 ST microarrays.</p> <p>Results</p> <p>Comparison of gene expression levels among <it>TMPRSS2-ERG </it>fusion-positive and negative tumors as well as benign samples demonstrated a distinct transcriptional program induced by the gene fusion event. Well-known biomarkers for prostate cancer detection like <it>CRISP3 </it>were found to be associated with the gene fusion status. WNT and TGF-β/BMP signaling pathways were significantly associated with genes upregulated in <it>TMPRSS2-ERG </it>fusion-positive tumors.</p> <p>Conclusions</p> <p>The <it>TMPRSS2-ERG </it>gene fusion results in the modulation of transcriptional patterns and cellular pathways with potential consequences for prostate cancer progression. Well-known biomarkers for prostate cancer detection were found to be associated with the gene fusion. Our results suggest that the fusion status should be considered in retrospective and future studies to assess biomarkers for prostate cancer detection, progression and targeted therapy.</p

ERG transcription factor is required to maintain high <i>TDRD1</i> expression.

<p>(A) <i>ERG</i> and <i>TDRD1</i> mRNA expression levels in VCaP cells measured 72 h after gene silencing with siRNAs. Three independent experiments were performed in triplicate. (B) ERG and TDRD1 protein expression in VCaP cells 72 h after gene silencing with siRNAs.</p

<i>TDRD1</i> does not control LINE1 activity in <i>TMPRSS2:ERG</i>-positive VCaP cells.

<p>(A) mRNA expression analysis of <i>PIWIL</i> genes in prostate cell lines by qRT-PCR. Testis RNA was used as a positive control. (B) mRNA expression analysis of LINE1 ORF2 in VCaP cells following 5 days of treatment with 5-aza-2′-deoxycytidine. (C) mRNA expression analysis of LINE1 ORF2 in VCaP cells following prolonged (8 days) <i>ERG</i> or <i>TDRD1</i> silencing. (D) Metabolic viability assay of VCaP cells treated with siRNAs. One (B), two (A) or three (C, D) independent experiments were performed in triplicate.</p

<i>TDRD1</i> promoter associated CpG island is hypomethylated in <i>TMPRSS2:ERG</i>-positive prostate cancer.

<p>(A) Analysis of <i>TDRD1</i> promoter methylation in prostate tumors by MeDIP-Seq. The values represent the average degree of DNA methylation of the 500-bp bins. (B) Correlation analysis of <i>TDRD1</i> promoter methylation and <i>TDRD1</i> mRNA expression in prostate cancer. The Spearman correlation coefficient is shown.</p

ERG-induced loss of epigenetic repression at the <i>TDRD1</i> promoter is a major mechanism of <i>TDRD1</i> overexpression.

<p>(A) DNA methylation analysis of the <i>TDRD1</i> promoter-associated CpG island in prostate cell lines by bisulfite sequencing. Average methylation level of the whole CpG island calculated from five sequenced colonies is shown (%). (B) Analysis of mRNA expression in LNCaP cells after treatment with the demethylating agent 5-aza-2′-deoxycytidine. Insert: analysis of protein expression in LNCaP cells after treatment with 5-aza-2′-deoxycytidine. 75µg of protein lysate from LNCaP cells was used per lane. (C) Analysis of mRNA expression in stable LNCaP clones overexpressing <i>ERG</i>. Two independent experiments were performed in triplicate. Insert: ERG expression analysis at 48 h in LNCaP clones by western blotting. (D) Bisulfite sequencing of the <i>TDRD1</i> promoter-associated CpG island in LNCaP cells 48 h after induction of ERG expression with doxycycline. (E) Bisulfite sequencing of the <i>TDRD1</i> promoter-associated CpG island 96 h after silencing of <i>ERG</i> in VCaP cells. The data shown in (D) and (E) are mean % of methylation of the entire CpG island calculated from 11–12 sequenced clones.</p

<i>TDRD1</i> is co-expressed with <i>ERG</i> but not with <i>ETV1</i> in prostate cancer.

<p>(A) Correlation analysis of mRNA levels measured by qRT-PCR in <i>TMPRSS2:ERG</i>-negative (ERG-, n = 30) and <i>TMPRSS2:ERG</i>-positive (ERG+, n = 17) prostate cancers as well as adjacent benign prostate tissue (n = 46). Pearson correlation coefficient is shown. (B) Analysis of mRNA expression in prostate cell lines by qRT-PCR. Two independent experiments were performed in triplicate. Human testis RNA was used as positive control for <i>TDRD1</i> expression. (C) Analysis of protein expression in prostate cell lines by western blotting. (D) Analysis of mRNA expression in hematopoietic cancer cell lines by qRT-PCR. Two independent experiments were performed in triplicate.</p