Androgen-induced TMPRSS2:ERG fusion in nonmalignant prostate epithelial cells

Abstract

Fusion genes play important roles in tumorigenesis. The identification of the high-frequency TMPRSS2 fusion with ERG and other ETS family genes in prostate cancer highlights the importance of fusion genes in solid tumor development and progression. However, the mechanisms leading to these fusions are unclear. We investigated whether androgen, through stimulating its receptor, could promote spatial genome reorganization and contribute to the generation of the TMPRSS2:ERG fusion. We show that treatment with androgen can induce the TMPRSS2:ERG fusion in both malignant and nonmalignant prostate epithelial cells. Although the fusion could be detected in malignant cells following 24-hour treatment, prolonged exposure to androgen was required to detect the fusion transcript in nonmalignant cells. We associated the fusion incidence with genetic factors, including androgen-induced gene proximity, androgen receptor exon1 CAG repeat length and expression of the PIWIL1 gene. This study demonstrates that fusions can be induced prior to malignant transformation and generation of the fusion is associated with both gene proximity and loss of the ability to prevent double-strand breaks.

Figure 1

TMPRSS2:ERG can be induced in non-malignant prostate epithelial cells following long-term DHT treatment. A, Nested RT-PCR amplification of induced TMPRSS2:ERG fusion product in non-malignant prostate cell lines PNT1a and PNT2 following 5 months treatment with 300 and 3000 nM DHT. B, Automated DNA sequencing of RT-PCR products confirms TMPRSS2:ERG fusion (exon1:exon4). A representative image from PNT1a cells is shown. C, Schematic representation of the TMPRSS2:ERG fusion event.

Figure 2

The TMPRSS2:ERG fusion cannot be detected in non-malignant prostate cell lines PNT1a and PNT2 following short-term DHT treatment. A, Nested RT-PCR was used to detect TMPRSS2:ERG fusion transcripts in the prostate cancer cell line LNCaP following 24 hours DHT treatment. No fusion product was detected in PNT1a, PNT2 and DU145 cells. B, Automated DNA sequencing of fusion-positive RT-PCR product confirm TMPRSS2:ERG fusion (exon1:exon4) in DHT treated LNCaP cells.

Figure 3

TMPRSS2 and ERG gene proximity was induced in non-malignant prostate cell lines PNT1a and PNT2 following DHT treatment. A, TMPRSS2 (red) and ERG (green) gene proximity was assessed by FISH in cells treated with and without DHT. * p<0.05; ** p<0.01. B, Representative FISH images.

Figure 4

TMPRSS2:ERG gene fusion frequency is associated with PIWIL1 expression. A, PIWIL1 expression, detected by Q-RT-PCR, was reduced in LNCaP as compared to PrEC, PNT1a and PNT2 cells and was undetectable in VCaP cells. B, PIWIL1 mRNA levels in LNCaP cells transfected with PIWIL1 expression construct. C, Nested RT-PCR detection of the TMPRSS2:ERG fusion transcript in LNCaP over-expressing PIWIL1. No fusion product was detected in PIWIL1 transfected cells. Ctl: untreated control cells; 300 nM: cells with long-term DHT treatment at 300 nM; 3000 nM: cells with long-term DHT treatment at 3000 nM; PIWIL1: PIWIL1 transfected cells; GFP: GFP transfected cells; DHT: 24-hour DHT treated cells.