Urinary Tract Infections Impair Adult Hippocampal Neurogenesis

Previous studies have suggested a link between urinary tract infections (UTIs) and cognitive impairment. One possible contributing factor for UTI-induced cognitive changes that has not yet been investigated is a potential alteration in hippocampal neurogenesis. In this study, we aim to investigate the effect of UTI on brain plasticity by specifically examining alterations in neurogenesis. Adult male Sprague Dawley rats received an intra-urethral injection of an Escherichia coli (E. coli) clinical isolate (108 CFU/mL). We found that rats with a UTI (CFU/mL ≥ 105) had reduced proliferation of neural stem cells (NSCs) at an early time point post infection (day 4) and neurogenesis at a later time point (day 34). This was associated with the decreased expression in mRNA of BDNF, NGF, and FGF2, and elevated expression of IL-1β in the hippocampus at 6 h post infection, but with no changes in optical intensity of the microglia and astrocytes. In addition, infected rats spent less time exploring a novel arm in the Y-maze test. Treatment with an anti-inflammatory drug did not revert the effect on NSCs, while treatment with antibiotics further decreased the basal level of their proliferation. This study presents novel findings on the impact of urinary tract infections on hippocampal neurogenesis that could be correlated with cognitive impairment

Self-renewing Pten-/- TP53-/- protospheres produce metastatic adenocarcinoma cell lines with multipotent progenitor activity.

Prostate cancers of luminal adenocarcinoma histology display a range of clinical behaviors. Although most prostate cancers are slow-growing and indolent, a proportion is aggressive, developing metastasis and resistance to androgen deprivation treatment. One hypothesis is that a portion of aggressive cancers initiate from stem-like, androgen-independent tumor-propagating cells. Here we demonstrate the in vitro creation of a mouse cell line, selected for growth as self-renewing stem/progenitor cells, which manifests many in vivo properties of aggressive prostate cancer. Normal mouse prostate epithelium containing floxed Pten and TP53 alleles was subjected to CRE-mediated deletion in vitro followed by serial propagation as protospheres. A polyclonal cell line was established from dissociated protospheres and subsequently a clonal daughter line was derived. Both lines demonstrate a mature luminal phenotype in vitro. The established lines contain a stable minor population of progenitor cells with protosphere-forming ability and multi-lineage differentiation capacity. Both lines formed orthotopic adenocarcinoma tumors with metastatic potential to lung. Intracardiac inoculation resulted in brain and lung metastasis, while intra-tibial injection induced osteoblastic bone formation, recapitulating the bone metastatic phenotype of human prostate cancer. The cells showed androgen receptor dependent growth in vitro. Importantly, in vivo, the deprivation of androgens from established orthotopic tumors resulted in tumor regression and eventually castration-resistant growth. These data suggest that transformed prostate progenitor cells preferentially differentiate toward luminal cells and recapitulate many characteristics of the human disease

Identification of Different Classes of Luminal Progenitor Cells within Prostate Tumors

Primary prostate cancer almost always has a luminal phenotype. However, little is known about the stem/progenitor properties of transformed cells within tumors. Using the aggressive Pten/Tp53-null mouse model of prostate cancer, we show that two classes of luminal progenitors exist within a tumor. Not only did tumors contain previously described multipotent progenitors, but also a major population of committed luminal progenitors. Luminal cells, sorted directly from tumors or grown as organoids, initiated tumors of adenocarcinoma or multilineage histological phenotypes, which is consistent with luminal and multipotent differentiation potentials, respectively. Moreover, using organoids we show that the ability of luminal-committed progenitors to self-renew is a tumor-specific property, absent in benign luminal cells. Finally, a significant fraction of luminal progenitors survived in vivo castration. In all, these data reveal two luminal tumor populations with different stem/progenitor cell capacities, providing insight into prostate cancer cells that initiate tumors and can influence treatment response

<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

Identification of Different Classes of Luminal Progenitor Cells within Prostate Tumors

Primary prostate cancer almost always has a luminal phenotype. However, little is known about the stem/progenitor properties of transformed cells within tumors. Using the aggressive Pten/Tp53-null mouse model of prostate cancer, we show that two classes of luminal progenitors exist within a tumor. Not only did tumors contain previously described multipotent progenitors, but also a major population of committed luminal progenitors. Luminal cells, sorted directly from tumors or grown as organoids, initiated tumors of adenocarcinoma or multilineage histological phenotypes, which is consistent with luminal and multipotent differentiation potentials, respectively. Moreover, using organoids we show that the ability of luminal-committed progenitors to self-renew is a tumor-specific property, absent in benign luminal cells. Finally, a significant fraction of luminal progenitors survived in vivo castration. In all, these data reveal two luminal tumor populations with different stem/progenitor cell capacities, providing insight into prostate cancer cells that initiate tumors and can influence treatment response

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

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