Preanalytical variables and performance of diagnostic RNA-based gene expression analysis in breast cancer

Prognostic multigene expression assays have become widely available to provide additional information to standard clinical parameters and to support clinicians in treatment decisions. In this study, we analyzed the impact of variations in tissue handling on the diagnostic EndoPredict test results. EndoPredict is a quantitative reverse transcription PCR assay conducted on RNA from formalin-fixed, paraffin-embedded (FFPE) tissue that predicts the likelihood of distant recurrence in patients with ER-positive/HER2-negative breast cancer. In this study, we performed a total of 138 EndoPredict assays to study the effects of preanalytical variables such as time to fixation, fixation time, tumor cell content, and section storage time on the EndoPredict test results. A time to fixation of up to 12 h and fixation of up to 5 days did not affect the results of the gene expression test. Paired samples of FFPE sections with tumor cell content ranging from 15 to 95 % and tumor-enriched samples showed a correlation coefficient of 0.97. Test results of tissue sections that have been stored for 12 months at +4 or +20 °C showed a correlation of 0.99 when compared to results of nonstored sections. In conclusion, preanalytical tissue handling is not a critical factor for diagnostic gene expression analysis with the EndoPredict assay. The test can therefore be easily integrated into the standard workflow of molecular pathology

Mutational analysis of p80 coilin indicates a functional interaction between coiled bodies and the nucleolus

Abstract. Coiled bodies are conserved subnuclear domains found in both plant and animal cells. They contain a subset of splicing snRNPs and several nucleolar antigens, including Nopp140 and fibrillarin. In addition, autoimmune patient sera have identified a coiled body specific protein, called p80 coilin. In this study we show that p80 coilin is ubiquitously expressed in human tissues. The full-length human p80 coilin protein correctly localizes in coiled bodies when exogenously expressed in HeLa cells using a transient transfection assay. Mutational analysis identifies separate domains in the p80 coilin protein that differentially affect its subnuclear localization. The data show that p80 coilin has a nuclear localization signal, but this is not sufficient to target the protein to coiled bodies. The results indicate that localizatio

Inhibition of protein dephosphorylation results in the accumulation of splicing snRNPs and coiled bodies within the nucleolus

Coiled bodies are conserved subnuclear organelles that contain splicing snRNPs, a subset of nucleolar antigens, and the autoantigen p80 coilin. Most nuclei contain one to five nucleoplasmic coiled bodies, often with one or more located at the nucleolar periphery. Here we show that exposure of mammalian cells to low levels of the specific Ser/Thr protein phosphatase inhibitor, okadaic acid, results in the accumulation of p80 coilin and splicing snRNPs within nucleoli. Mutation of a single serine residue in p80 coilin to aspartate (S202D) also causes coiled bodies and splicing snRNPs to localize within nucleoli when the mutant is transiently transfected and expressed in HeLa cells. Neither okadaic acid nor the S202D coilin mutant causes nucleolar accumulation of serine-arginine domain proteins. These data indicate that protein dephosphorylation is required to allow normal formation of nucleoplasmic coiled bodies and point to p80 coilin as a substrate whose phosphorylation state may regulate snRNP-nucleolar interactions. The data are consistent with a trafficking mechanism whereby splicing snRNPs cycle through the nucleolus. (C) 1997 Academic Press</p

Keap1-Independent Regulation of Nrf2 Activity by Protein Acetylation and a BET Bromodomain Protein.

Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. We identified the sole member of the BET protein family in Drosophila, Fs(1)h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1)h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1)h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1)h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation

Fs(1)h negatively regulates CncC signaling.

<p>(A) The <i>Drosophila fs(1)h</i> gene encodes two protein isoforms: the 120kD Fs(1)h-S and the 210kD Fs(1)h-L. Both isoforms contain two bromodomains (BD) and an extraterminal (ET) domain. In addition, Fs(1)h-L carries a unique C-terminal motif (CTM). (B) dsRNA-mediated knock down of Fs(1)h (3.6 fold, P<0.05), like Keap1 knock down (3.9 fold, P<0.01), increases the activity of a transiently transfected ARE-fluc reporter in S2 cells. In both cases, this stimulatory effect is suppressed by CncC knock down. (C) RT-qPCR experiments show that the CncC target genes <i>gstD1</i> (P<0.0001), <i>gclC</i> (P<0.001) and <i>keap1</i> (P<0.05) are activated upon RU486-induced-knock down of Fs(1)h under the control of tub-GS-Gal4 driver. Measurements of transcript abundance levels were normalized to <i>act5c</i> transcript levels. Fold activation relative to the mRNA levels in mock treated flies is shown. The error bars indicate standard deviation of 3 biological replicates (flies collected from separate vials). (D) Knock down of Fs(1)h-L alone using a specific dsRNA targeting the CTM region was sufficient to induce ARE-fluc activity in S2 cells (3 fold, P<0.01). However, knock down of Fs(1)h-S alone with dsRNA targeting its 3’ UTR did not induce ARE-fluc activity. Error bars in panels B, C and D signify standard deviation of 3 biological replicates.</p

Bromodomains mediate the inhibition of CncC by Fs(1)h.

<p>(A) S2 cells transfected with the ARE-fluc reporter plasmid were treated with 1μM JQ1 for 24 hrs, which stimulated ARE-fluc activity (6.5 fold, P<0.001). This increase was strongly diminished after knock down of CncC (JQ1 mediated induction is 3.2 fold, P<0.001) or MafS (JQ1 mediated induction is 4.2 fold, P<0.002). Combined knock down of MafS and CncC almost completely eliminates the JQ1 effect (JQ1 mediated induction is 1.6 fold, P>0.05). The diagram shows the fold change in luciferase activity relative to controls. Error bars indicate standard deviation of 3 biological replicates. (B) 5 day old adult ARE-GFP flies were maintained on food containing 0.25 mM JQ1 for 2 days. Fluorescence images of adult flies showed a strong induction of ARE-GFP reporter activity by JQ1. Two randomly chosen JQ1-treated and two solvent-treated female flies are shown. (C) Co-immuno-precipitation of endogenous Fs(1)h-L with over-expressed CncC-Flag in S2 cells. S2 cells were transfected with either actin-Gal4 plasmid alone (lane 1) or with actin-Gal4 and UAS-CncC-Flag plasmids (lanes 2–5). The cells over-expressing CncC-Flag were treated either with the HDAC inhibitor LBH589 (500nM) and/or JQ1 (10μM) as indicated, or with 0.01% DMSO (vehicle) for 6 hours before they were processed for immuno-precipitation. 10μM JQ1 was also added to the lysate from cells treated with JQ1 to assess the effect of JQ1 on Fs(1)h-CncC interaction. Immuno-precipitation was performed using anti-Flag antibody followed by immuno-blotting with anti-Fs(1)h-L antibody. The acetylation status of CncC in the same immuno-precipitates was examined in western blots using an antibody against acetylated lysine.</p

Fs(1)h-L cell-autonomously inhibits CncC activity and affects oxidative stress resistance in adult flies.

<p>(A) Ubiquitous knock down of Fs(1)h (in the v51227 RNAi line) in adult flies, using the RU486-inducible tub-GS-Gal4 driver stimulates ARE-GFP reporter activity in most tissues. Two RU486-treated and two mock-treated females are shown in this panel and in panel C. The same flies are shown under UV-illumination to visualize GFP fluorescence and under white light. (B) Knock down of Fs(1)h in actin-flipout-Gal4 clones increased ARE-GFP activity in a cell-autonomous manner in the crop of adult <i>Drosophila</i> gut. In the cells in which Fs(1)h expression is knocked down (marked by the expression of RFP, red) ARE-GFP reporter activity (green) is increased. (C) RU486-induced over-expression of Fs(1)h from the EP-fs(1)h allele reduces oltipraz-stimulated ARE-GFP reporter activity in the whole body. Similar effects are seen after ubiquitous over-expression of Fs(1)h-L from a UAS construct (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006072#pgen.1006072.s002" target="_blank">S2 Fig</a>). (D) Fs(1)h-L over-expression in actin-flipout-Gal4 clones (labeled by RFP, red), cell- autonomously reduces ARE-GFP activity in the ejaculatory bulb of adult males. (E) Ubiquitous Fs(1)h knock-down in adult <i>Drosophila</i> by inducible expression of a UAS-Fs(1)h^RNAi transgene under the control of the tub-GS-Gal4 driver increases oxidative stress resistance. Survival after exposure to 20μM DEM was recorded and the data were analyzed by Mantel-Cox log-rank test. Female flies incubated on RU486 food, showed significantly increased resistance to DEM (P value <0.001) compared to those on control food. A similar effect was observed in males (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006072#pgen.1006072.s003" target="_blank">S3 Fig</a>). The standard deviations of percent survival among biological replicates in this and other stress sensitivity assays are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006072#pgen.1006072.s006" target="_blank">S1 Document</a>. (F) Fs(1)h over-expression increases stress sensitivity. Fs(1)h was over-expressed from the EP-fs(1)h allele in female flies by exposing them to food containing 300μM RU486 for 4 days. Lethality after exposure to 20μM DEM was recorded and analyzed by Mantel-Cox log-rank test. The flies that were kept on RU486 food, showed significantly increased sensitivity to DEM (P value < 0.0001) compared to those on control food. The same experiment was also conducted with males and produced qualitatively the same result (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006072#pgen.1006072.s003" target="_blank">S3 Fig</a>).</p