Neoplastic lesions in <i>TgK18GT₁₂₁; β-actin Cre</i> mice.

<p>Hyperplasia was observed in some tissues (ovary and prostate: indicated by arrows). All mice were euthanized at 1–7 months of age due to life-threatening thoracic pressure caused by enlarged thymuses. All samples were stained with H&E.</p

Proliferation in T₁₂₁-expressing <i>TgK19GT₁₂₁;</i><i>β-actin Cre</i> tissues.

<p>Proliferation was assessed by Ki-67 IHC (brown) in tissues of 2 months old mice. Arrows indicate ovarian surface epithelial cells (OSECs) positive for Ki-67. Scale bar = 50 µM.</p

Scheme of transgene constructs and copy number.

<p>A. Transgene cassette: An eGFP stop cassette was flanked by loxP sites and the T₁₂₁ gene was placed downstream. B. Keratin bacterial artificial chromosome (BAC) transgene constructs: a transgene cassette (A) was inserted upstream of the start ATG codon in exon 1 of K18 (derived from mouse chromosome (Chr.) 15) and of K19 (derived from mouse Chr 11) in BACs using recombineering. The insertion site for the loxP-eGFP-stop-loxP-T₁₂₁ cassette is indicated by a dashed “V”. Representative genes downstream of keratin genes are indicated in boxes. Solid black bars indicate exons (E). C. Transgene copy number per diploid genome was determined by qPCR.</p

Proliferation in T₁₂₁-expressing <i>TgK18GT₁₂₁;</i><i> </i><i>β-actin Cre</i> tissues.

<p>Proliferation was assessed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080459#pone-0080459-g005" target="_blank">Figure 5</a> in 2 month old mice. Similar proliferation levels were observed in small intestine, colon, stomach and ovarian follicle as that of wildtype mice shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080459#pone.0080459.s008" target="_blank">Figure S8</a>. Black arrows indicate Ki-67 positive cells. Scale bar = 50 µM.</p

Summary of histopathological findings in <i>TgK19GT₁₂₁; β-actin Cre</i> and <i>TgK18GT₁₂₁; β-actin Cre</i> mice.

*<p>Average age of <i>TgK19GT₁₂₁; β-actin Cre</i> mice analyzed here was 123 days (2–6 months, mainly line 43) and <i>TgK18GT₁₂₁; β-actin Cre</i> mice 130 days (1–6 months, both lines). Three <i>TgK19GT₁₂₁; β-actin Cre</i> mice were euthanized due to kidney hydronephrosis at 2 months of age. All <i>TgK18GT₁₂₁; β-actin Cre</i> mice were euthanized due to thymic masses at 1–6 months of age.</p>†<p>The onset and extent of lesions were different between <i>TgK19GT₁₂₁; β-actin Cre</i> and <i>TgK18GT₁₂₁; β-actin Cre</i> mice (see text for details).</p>#<p>multifocal hyperplasia. m: male; f: female; N.D.: not determined.</p

Proliferation rates in tissues of <i>TgK19GT₁₂₁;</i><i> </i><i>β-actin Cre</i> and <i>TgK18GT₁₂₁;</i><i> </i><i>β-actin Cre</i> mice.

<p>Proliferation rates of pancreas, mammary gland, ovary, prostate (dorsolateral lobe), and bladder were quantified on tissue sections stained with Ki-67. *Significantly different from WT control (p<0.001); #significantly different between <i>TgK19GT₁₂₁; β-actin Cre and TgK18GT₁₂₁; β-actin Cre</i> (p = 0.007); @ significantly different between <i>TgK18GT₁₂₁; β-actin Cre</i> and WT (p = 0.043).</p

Neoplastic lesions in <i>TgK19GT₁₂₁;</i><i>β-actin Cre</i> mice.

<p>Hyperplasia was observed in most tissues (lung and colon: indicated by black arrows). Multifocal ductal hyperplasia was observed in pancreas, as well as mouse prostate intraepithelial neoplasia (mPIN) in prostate, and hydronephrosis as indicated by white arrows in kidney. All samples were stained with H&E.</p

Summary of neoplastic lesions developed in tamoxifen-induced <i>TgK19GT₁₂₁-34;K19CreER</i> mice.

<p>Males developed neoplastic lesions at 9 months post induction (PI), while females at 14 months PI except one at 8 months PI (*labeled). mo: month.</p

<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