<em>TMPRSS2-</em> Driven <em>ERG</em> Expression <em>In Vivo</em> Increases Self-Renewal and Maintains Expression in a Castration Resistant Subpopulation

<div><p>Genomic rearrangements commonly occur in many types of cancers and often initiate or alter the progression of disease. Here we describe an in vivo mouse model that recapitulates the most frequent rearrangement in prostate cancer, the fusion of the promoter region of <em>TMPRSS2</em> with the coding region of the transcription factor, <em>ERG</em>. A recombinant bacterial artificial chromosome including an extended <em>TMPRSS2</em> promoter driving genomic <em>ERG</em> was constructed and used for transgenesis in mice. <em>TMPRSS2-ERG</em> expression was evaluated in tissue sections and FACS-fractionated prostate cell populations. In addition to the anticipated expression in luminal cells, <em>TMPRSS2-ERG</em> was similarly expressed in the Sca-1^hi/EpCAM⁺ basal/progenitor fraction, where expanded numbers of clonogenic self-renewing progenitors were found, as assayed by in vitro sphere formation. These clonogenic cells increased intrinsic self renewal in subsequent generations. In addition, ERG dependent self-renewal and invasion in vitro was demonstrated in prostate cell lines derived from the model. Clinical studies have suggested that the <em>TMPRSS2-ERG</em> translocation occurs early in prostate cancer development. In the model described here, the presence of the <em>TMPRSS2-ERG</em> fusion alone was not transforming but synergized with heterozygous <em>Pten</em> deletion to promote PIN. Taken together, these data suggest that one function of <em>TMPRSS2-ERG</em> is the expansion of self-renewing cells, which may serve as targets for subsequent mutations. Primary prostate epithelial cells demonstrated increased post transcriptional turnover of ERG compared to the TMPRSS2-ERG positive VCaP cell line, originally isolated from a prostate cancer metastasis. Finally, we determined that <em>TMPRSS2-ERG</em> expression occurred in both castration-sensitive and resistant prostate epithelial subpopulations, suggesting the existence of androgen-independent mechanisms of TMPRSS2 expression in prostate epithelium.</p> </div

Expression of TMPRSS2-ERG in subpopulations of primary prostate epithelial cells fractionated by FACS.

<p>(A) Scatter plot of lineage negative (Lin-) prostate populations labeled for Sca-1 and EpCAM. The gated regions for the four isolated populations are shown. Upper left quadrant: Sca-1+; lower left quadrant: Sca-1/EpCAM-; upper right quadrant: Sca-1hi/EpCAM+; lower right quadrant: EpCAM+. (B) Representative examples are shown for QRT-PCR determined expression in WT and A5 RNA samples of the various fractions. Expression values were normalized to <i>Gapdh</i>, and the highest resulting expression value for each primer pair was set to 1. (C) Isoform-specific expression of ERG is shown for ERG fusion, ERG8, and ERG exon 16 using primer pairs b/d, i/j, and g/h, respectively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041668#pone-0041668-g001" target="_blank">Figure 1A</a>).</p

Description of lesion development when TMPRSS2-ERG is crossed with <i>Nkx3.1^-/-</i> and <i>Pten</i>^+/- transgenic lines.

<p>The numbers of mice displaying the indicated lesions relative to the number of mice analyzed is shown. The values in parentheses correspond to the average number of lesions per prostate where a significant increase in mPIN was observed in A5/<i>Pten</i>^+/- compared to <i>Pten</i>^+/- at 28 weeks.</p>a<p><i>p</i>≤0.05.</p>b<p>Numbers of lesions per animal ranged from 0–17<i>^b p</i> = 0.15.</p

Expression of TMPRSS2 ERG in basal and luminal prostate cells.

<p>(A) Images 1–8. WT (1–4) and A5 (5–8) prostate sections stained with Dapi (1&5) and ERG Ab 2805 (2&6). Composite images (3&7) show increased ERG staining in transgenic compared to WT. Enlarged regions (4&8) show ERG positive endothelial cells found in WT and transgenic tissue, indicated by triangle symbol, and ERG positive epithelial cells, present in A5 tissue, indicated by arrowheads. (B) Images 1–8. WT (1–4) and A5 (5–8) prostate sections stained with Dapi (1&5), ERG Ab 2805 (2&6) and KRT8 (3&7). Composite images (4&8) show KRT8/ERG positive luminal cells. (C) Images 1–10. WT (1–5) and A5 (6–10) prostate sections stained with Dapi (1&6), ERG Ab 2805 (2&7) and TP63 (3&8) where arrows indicate positive staining. Composite images (4&9) and enlarged regions (5&10) show ERG positive basal cells are present in A5 prostate. Twenty three percent of TP63⁺cells co-stained for ERG (5 fields). Symbols correspond to the following cells/stains; Triangle symbol, ERG positive endothelial cell; Arrows, TP63 positive basal cells where absence of an asterix corresponds to an ERG positive cell and presence corresponds to an ERG negative basal cell; Arrowheads, ERG positive luminal cells. For all images, scale bars = 20 um.</p

Characterization of ERG expression within the transgenic lines.

<p>(A) Western blot of ERG expression in WT and fusion prostate organoid cultures. * denotes non-specific signal. (B) Quantification of ERG expression level within organoid cultures (n = 3) using primers e/f and ERG FAM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041668#pone.0041668.s004" target="_blank">Table S1</a>). Comparison to VCaP (two independent replicates) is shown where copies/ng total RNA were derived using a standard curve for ERG copy number and samples were normalization to Gapdh. (C) Western blot comparing relative Erg protein expression in the VCaP cell line (2 ug of protein loaded) and organoid cultures (20 ug protein loaded per sample), using Ab ERG 5115.</p

The introduced TMPRSS2 promoter binds AR in primary epithelial cells.

<p>(A) Schematic of the introduced human TMPRSS2 genomic region present within the BAC. The location relative to exon 1 (+1) of androgen response elements (ARE’s) that were bound by AR as presented in (B) are shown. (B) ChIP analysis for bound AR of fusion prostatic tissue (n = 2 for each line) <i>Fkbp5</i> and <i>ActB</i> are positive and negative controls, respectively, for AR binding. Fold enrichment corresponds to the QPCR signal in AR antibody samples relative to rabbit IgG controls. Error bars correspond to ± s.d.</p

<i>TMPRSS2</i> is expressed in castration-sensitive and resistant populations and is not responsive to supraphysiological concentrations of androgen.

<p>(A) QRT-PCR expression analysis of ERG fusion transcripts using primer pairs b/d (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041668#pone-0041668-g001" target="_blank">Figure 1A</a>) in A5 transgenic prostates isolated from uncastrated (n = 8), castrated (n = 9), and castrated animals with subsequent androgen supplementation, where tissue was harvested at days 3 (n = 3) or 7 (n = 3) post pellet implantation. (B) QRT-PCR expression analysis of lineage markers and known AR regulated genes from the samples described in (A). Error bars correspond to ± s.d, (C-D) QRT-PCR analysis of RNA isolated from prostate tissue with (squares) or without (circles) androgen pellet supplementation for 3 days was performed, and the levels of expression following normalization to <i>Gapdh</i> are shown. Symbols represent individual animals. Expression of (C) <i>Fkbp5</i> and (D) the TMPRSS2-ERG fusion, assessed using primers b/d (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041668#pone-0041668-g001" target="_blank">Figure 1A</a>), are shown. ***P<0.001, **P<0.01, *P<0.05.</p