Role of the TMPRSS2-ERG gene fusion in prostate cancer

Abstract

TMPRSS2-ERG gene fusions are the predominant molecular subtype of prostate cancer. Here, we explored the role of TMPRSS2-ERG gene fusion product using in vitro and in vivo model systems. Transgenic mice expressing the ERG gene fusion product under androgen-regulation develop mouse prostatic intraepithelial neoplasia (PIN), a precursor lesion of prostate cancer. Introduction of the ERG gene fusion product into primary or immortalized benign prostate epithelial cells induced an invasion-associated transcriptional program but did not increase cellular proliferation or anchorage-independent growth. These results suggest that TMPRSS2-ERG may not be sufficient for transformation in the absence of secondary molecular lesions. Transcriptional profiling of ERG knockdown in the TMPPRSS2-ERG-positive prostate cancer cell line VCaP revealed decreased expression of genes over-expressed in prostate cancer versus PIN and genes overexpressed in ETS-positive versus -negative prostate cancers in addition to inhibiting invasion. ERG knockdown in VCaP cells also induced a transcriptional program consistent with prostate differentiation. Importantly, VCaP cells and benign prostate cells overexpressing ERG directly engage components of the plasminogen activation pathway to mediate cellular invasion, potentially representing a downstream ETS target susceptible to therapeutic intervention. Our results support previous work suggesting that TMPRSS2-ERG fusions mediate invasion, consistent with the defining histologic distinction between PIN and prostate cancer.

Figure 1

Transgenic mice recapitulating TMPRSS2-ERG in the prostate develop mPIN. (a) To recapitulate TMPRSS2-ERG in vivo, we generated transgenic mice over-expressing the ERG gene fusion product (exons 2 through the reported stop codon; 1533 of NM_182918.2, C-terminal 3X-FLAG epitope tag) with a bovine growth hormone polyA signal (PA-BGH) under the control of the enhanced probasin promoter (ARR2Pb). Mice were sacrificed at 12 to 14 weeks or >20 weeks, and mouse prostatic intraepithelial neoplasia (mPIN) was observed in 4 of 11 ARR2Pb-ERG mice as described in Table W1. Benign epithelia and areas of mPIN are indicated by yellow and black arrows, respectively. (b–d) Hematoxylin and eosin staining of ARR2Pb-ERG prostates for morphologic assessment. Consistent with the focality of mPIN, (b) benign glands and (c and d) mPIN were observed in the ventral prostate (VP) of ARR2Pb-ERG mice. Original magnification: (b) ×400, (c) ×200, and (d) inset showing area of mPIN with macronucleoli, ×400.

Figure 2

Over-expression of ERG in RWPE cells increases invasion through the plasminogen activator pathway. (a) To recapitulate TMPRSS2-ERG in vitro, we generated adenoviruses and lentiviruses expressing the ERG gene fusion product (exons 2 through the reported stop codon). (b and c) Infected (b) RWPE and (c) PrEC cells as indicated were assayed for invasion through a modified basement membrane. Photomicrographs of invaded cells are shown below. (d) RWPE-ERG and RWPE-GUS (control vector) cells were profiled on Agilent Whole Genome microarrays and expression signatures were loaded into the Oncomine Concept Map. Molecular concept map analysis of the over-expressed in RWPE-ERG compared to RWPE-GUS signature (ringed yellow node). Each node represents a molecular concept, or set of biologically related genes. The node size is proportional to the number of genes in the concept. The concept color indicates the concept type according to the legend. Each edge represents a significant enrichment (P < .005). (e) qPCR confirmation of increased expression of genes involved in invasion. The amount of the indicated gene (normalized to the average of GAPDH and HMBS) in RWPE-GUS (white) and RWPE-ERG (black) is shown. Inset shows immunoblot confirmation of increased expression of PLAU and MMP3 in RWPE-ERG cells. (f) Chromatin immunoprecipitation shows enrichment of ERG binding to the proximal promoters of PLAU and MMP3 compared to IgG control. The promoter of KIAA0089 was used as a negative control. (g) RWPE-ERG cells were treated with PLAU inhibitors amiloride or ectopic PAI-1, MMP inhibitors (including the pan-MMP inhibitor GM-6001), or the EWS:FLI inhibitor ARA-C (EWS:FLI inhibitor) as indicated and assayed for invasion as in c. (h) RWPE-ERG cells were treated with transfection reagent alone (untreated), or transfected with nontargeting, PLAU or PLAT siRNA as indicated and assayed for invasion through a modified basement membrane. For all invasion assays, mean (n = 3) ± SEM are shown; *P < .05.31

Figure 3

Knockdown of ERG in VCaP cells attenuates a transcriptional program over-expressed in TMPRSS2-ETS-positive prostate cancers. (a) SiRNA knockdown of ERG in the TMPRSS2-ERG-positive prostate cancer cell line VCaP. VCaP cells were treated with transfection reagent alone (untreated), or transfected with nontargeting or ERG siRNA (VCaP-siERG) as indicated. ERG knockdown was confirmed by immunoblot analysis. (b) VCaP cells as indicated were assayed for invasion through a modified basement membrane. (c) VCaP-siERG and VCaP cells treated with nontargeting siRNA were profiled and a molecular concept map of the under-expressed in VCaP-siERG signature (ringed yellow node) was generated. Each edge represents a significant enrichment (P < .001). Blue edges indicate enrichments with in vivo ETS-positive versus negative prostate cancer signatures. (d) Chromatin immunoprecipitation identifies PLAT and PLAU as direct targets of ERG in VCaP cells, by enrichment of ERG binding to the proximal promoters of PLAT and PLAU compared to IgG control. The promoter of KIAA0089 was used as a negative control. (e) VCaP cells were treated with the indicated inhibitors (as in Figure 2g) and assessed for invasion. (f) VCaP cells were treated with transfection reagent alone (untreated), or transfected with nontargeting, PLAU or PLAT siRNA as indicated and assayed for invasion. For all invasion assays, mean (n = 3) ± SEM are shown; *P < .05.

Figure 4

ERG knockdown in VCaP cells derepresses a transcriptional program associated with normal prostatic epithelial cell differentiation. (a) VCaP-siERG and VCaP cells treated with nontargeting siRNA were profiled and a molecular concept map of the over-expressed in VCaP-siERG signature (ringed yellow node) was generated. Each edge represents a significant enrichment (P < .001). Blue edges indicate enrichments with in vivo ETS-positive versus -negative prostate cancer signatures. (b) Overlay map identifying genes present (red cells), including KLK3 (PSA), across multiple concepts in the over-expressed in VCaP-siERG enrichment network (indicated by number). (c) qPCR confirmation of increased expression in VCaP-siERG cells (black) compared to VCaP-NT cells (white) of transcripts strongly expressed in prostatic epithelial cells. (d) Analysis of prostate cell type specificity using a microarray data set profiling magnetically sorted prostate cell populations. Mean RMA normalized fluorescent intensities (n = 5 ± SEM) are shown. *P < .05, for all pairwise t tests involving luminal cells.