Role of TMPRSS2-ERG gene fusion in negative regulation of PSMA expression - PubMed (original) (raw)

Role of TMPRSS2-ERG gene fusion in negative regulation of PSMA expression

Lihong Yin et al. PLoS One. 2011.

Abstract

Prostate specific membrane antigen (PSMA) is overexpressed in prostatic adenocarcinoma (CaP), and its expression is negatively regulated by androgen stimulation. However, it is still unclear which factors are involved in this downregulation. TMPRSS2-ERG fusion is the most common known gene rearrangement in prostate carcinoma. Androgen stimulation can increase expression of the TMPRSS2-ERG fusion in fusion positive prostate cancer cells. The purpose of this investigation is to determine whether PSMA expression can be regulated by the TMPRSS2-ERG gene fusion. We employed two PSMA positive cell lines: VCaP cells, which harbor TMPRSS2-ERG fusion, and LNCaP cells, which lack the fusion. After 24 hours of androgen treatment, TMPRSS2-ERG mRNA level was increased in VCaP cells. PSMA mRNA level was dramatically decreased in VCaP cells, while it only has moderate change in LNCaP cells. Treatment with the androgen antagonist flutamide partially restored PSMA expression in androgen-treated VCaP cells. Knocking down ERG by siRNA in VCaP cells enhances PSMA expression both in the presence and absence of synthetic androgen R1881. Overexpressing TMPRSS2-ERG fusions in LNCaP cells downregulated PSMA both in the presence or absence of R1881, while overexpressing wild type ERG did not. Using PSMA-based luciferase reporter assays, we found TMPRSS2-ERG fusion can inhibit PSMA activity at the transcriptional level. Our data indicated that downregulation of PSMA in androgen-treated VCaP cells appears partially mediated by TMPRSS2-ERG gene fusion.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Inhibition of PSMA by androgen in VCaP cells.

A, Expression levels of TMPRSS2-ERG in VCaP cells by real-time PCR, normalized to PGD mRNA level. ERG protein level was measured by western blot after treatment with 5 nM of R1881 or 10 µM of antagonist flutamide for 1 day. B, PSMA expression was detected by real-time PCR in VCaP cells, normalized to PGD. (Cells were treated the same as in Fig 1A). C, PSMA protein levels were checked by western blot in VCaP cells treated with 5 nM of R1881 for 0–5 days. For real-time PCR, cells were treated with vehicle or androgen antagonist flutamide for 2 hours prior to the treatment of the synthetic androgen R1881 for 24 hours. Experiments were done in triplicate.

Figure 2

Figure 2. Knocking down ERG by siRNA enhances PSMA expression in VCaP cells.

A, Cells were transfected with different ERG siRNA by oligofectamine for 48 hours. ERG expression levels were tested by quantitative real-time PCR, normalized to PGD mRNA level. B, Real-time PCR for PSMA expression in ERG knockdown VCaP cells. Cells were treated with or without R1881 for 24 hours after 48 hours treatment of siRNA. C, Western blot for ERG and PSMA expression in ERG knockdown VCaP cells. Cells were harvested 72 hours post siRNA transfection.

Figure 3

Figure 3. Overexpression of TMPRSS2-ERG fusions decreased PSMA expression in LNCaP cells.

A, PSMA expression was detected by real-time PCR in LNCaP cells, normalized to PGD mRNA level. Cells were treated with vehicle DMSO or androgen antagonist flutamide for 2 hours prior to the treatment of the synthetic androgen R1881 for 24 hours. B, TMPRSS2-ERG fusion protein levels were checked by Western blot using anti-V5 antibody. LNCaP cells were transfected with fusion type III, III+72, VI, VI+72, or empty vector for 48 hours. C, Real-time PCR detected PSMA mRNA level in TMPRSS2-ERG fusion-transfected LNCaP cells. Cells were transfected with fusions or empty vector for 48 hours, then treated with or without R1881 for 24 hours. D, ERG expression level in ERG-transfected LNCaP cells by western blot. Cells were transfected with full length ERG for 48 hours. E, PSMA mRNA level in ERG overexpressing LNCaP cell. Cells were treated with or without R1881 for 24 hours post 48 hours of transfection. Experiments were done in triplicate.

Figure 4

Figure 4. PSMA luciferase activity in VCaP or LNCaP cells.

A, Graphic prediction of ETS transcription factor binding site on PSMA promoter and recruitment of ERG to PSMA promoter detected by ChIP assay. B, PSMA luciferase activity in VCaP cells. VCaP cells were transfected with PSM-Luc and Renilla-Luc luciferase reporter genes for 24 hours, then cells were treated with R1881 and androgen antagonist flutamide for another 24 hours. C and D, PSMA luciferase activity in LNCaP and C4-2 cells. LNCaP and C4-2 cells were co-transfected with PSM-Luc, Renilla-Luc, and TMPRSS2-ERG fusions, type III, III+72, VI, VI+72. Twenty-four hours post transfection, cells were cultured in the presence or absence of R1881 for another 24 hours.

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