Identification of Differential Gene Expression in Brassica rapa Nectaries through Expressed Sequence Tag Analysis

BACKGROUND: Nectaries are the floral organs responsible for the synthesis and secretion of nectar. Despite their central roles in pollination biology, very little is understood about the molecular mechanisms underlying nectar production. This project was undertaken to identify genes potentially involved in mediating nectary form and function in Brassica rapa. METHODOLOGY AND PRINCIPAL FINDINGS: Four cDNA libraries were created using RNA isolated from the median and lateral nectaries of B. rapa flowers, with one normalized and one non-normalized library being generated from each tissue. Approximately 3,000 clones from each library were randomly sequenced from the 5' end to generate a total of 11,101 high quality expressed sequence tags (ESTs). Sequence assembly of all ESTs together allowed the identification of 1,453 contigs and 4,403 singleton sequences, with the Basic Localized Alignment Search Tool (BLAST) being used to identify 4,138 presumptive orthologs to Arabidopsis thaliana genes. Several genes differentially expressed between median and lateral nectaries were initially identified based upon the number of BLAST hits represented by independent ESTs, and later confirmed via reverse transcription polymerase chain reaction (RT PCR). RT PCR was also used to verify the expression patterns of eight putative orthologs to known Arabidopsis nectary-enriched genes. CONCLUSIONS/SIGNIFICANCE: This work provided a snapshot of gene expression in actively secreting B. rapa nectaries, and also allowed the identification of differential gene expression between median and lateral nectaries. Moreover, 207 orthologs to known nectary-enriched genes from Arabidopsis were identified through this analysis. The results suggest that genes involved in nectar production are conserved amongst the Brassicaceae, and also supply clones and sequence information that can be used to probe nectary function in B. rapa

Activation of PPARγ in Myeloid Cells Promotes Lung Cancer Progression and Metastasis

Activation of peroxisome proliferator-activated receptor-γ (PPARγ) inhibits growth of cancer cells including non-small cell lung cancer (NSCLC). Clinically, use of thiazolidinediones, which are pharmacological activators of PPARγ is associated with a lower risk of developing lung cancer. However, the role of this pathway in lung cancer metastasis has not been examined well. The systemic effect of pioglitazone was examined in two models of lung cancer metastasis in immune-competent mice. In an orthotopic model, murine lung cancer cells implanted into the lungs of syngeneic mice metastasized to the liver and brain. As a second model, cancer cells injected subcutaneously metastasized to the lung. In both models systemic administration of pioglitazone increased the rate of metastasis. Examination of tissues from the orthotopic model demonstrated increased numbers of arginase I-positive macrophages in tumors from pioglitazone-treated animals. In co-culture experiments of cancer cells with bone marrow-derived macrophages, pioglitazone promoted arginase I expression in macrophages and this was dependent on the expression of PPARγ in the macrophages. To assess the contribution of PPARγ in macrophages to cancer progression, experiments were performed in bone marrow-transplanted animals receiving bone marrow from Lys-M-Cre+/PPARγflox/flox mice, in which PPARγ is deleted specifically in myeloid cells (PPARγ-Macneg), or control PPARγflox/flox mice. In both models, mice receiving PPARγ-Macneg bone marrow had a marked decrease in secondary tumors which was not significantly altered by treatment with pioglitazone. This was associated with decreased numbers of arginase I-positive cells in the lung. These data support a model in which activation of PPARγ may have opposing effects on tumor progression, with anti-tumorigenic effects on cancer cells, but pro-tumorigenic effects on cells of the microenvironment, specifically myeloid cells

Variable Expression of PIK3R3 and PTEN in Ewing Sarcoma Impacts Oncogenic Phenotypes

<div><p>Ewing Sarcoma is an aggressive malignancy of bone and soft tissue affecting children and young adults. Ewing Sarcoma is driven by EWS/Ets fusion oncoproteins, which cause widespread alterations in gene expression in the cell. Dysregulation of receptor tyrosine kinase signaling, particularly involving IGF-1R, also plays an important role in Ewing Sarcoma pathogenesis. However, the basis of this dysregulation, including the relative contribution of EWS/Ets-dependent and independent mechanisms, is not well understood. In the present study, we identify variable expression of two modifiers of PI3K signaling activity, PIK3R3 and PTEN, in Ewing Sarcoma, and examine the consequences of this on PI3K pathway regulation and oncogenic phenotypes. Our findings indicate that PIK3R3 plays a growth-promotional role in Ewing Sarcoma, but suggest that this role is not strictly dependent on regulation of PI3K pathway activity. We further show that expression of PTEN, a well-established, potent tumor suppressor, is lost in a subset of Ewing Sarcomas, and that this loss strongly correlates with high baseline PI3K pathway activity in cell lines. In support of functional importance of PTEN loss in Ewing Sarcoma, we show that re-introduction of PTEN into two different PTEN-negative Ewing Sarcoma cell lines results in downregulation of PI3K pathway activity, and sensitization to the IGF-1R small molecule inhibitor OSI-906. Our findings also suggest that PTEN levels may contribute to sensitivity of Ewing Sarcoma cells to the microtubule inhibitor vincristine, a relevant chemotherapeutic agent in this cancer. Our studies thus identify PIK3R3 and PTEN as modifiers of oncogenic phenotypes in Ewing Sarcoma, with potential clinical implications.</p></div

PIK3R3 depletion results in inhibition of clonogenic and anchorage-independent growth of Ewing Sarcoma cells.

<p>(A) Stable depletion of PIK3R3 protein in Ewing Sarcoma SK-ES-1 cells was achieved using lentiviral delivery of two different targeting shRNAs (PIK3R3-sh1 and 2, described in Methods; shControl: cells expressing non-targeting scrambled sequence RNA, also described in Methods). PIK3R3 RNA levels were determined by qRT-PCR, as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116895#pone.0116895.ref019" target="_blank">19</a>], using specific primers to PIK3R3, and U6 as an endogenous control. PIK3R3 protein levels were determined by immunoblotting with PIK3R3 antibody, normalized to tubulin and quantified by densitometry. Mean and standard deviation are plotted at top, and a representative immunoblot is shown below. Mean values in the control groups are set to 1; *p<0.05, relative to corresponding control, using the student t-test. (B) Colony growth of control and PIK3R3-depleted SK-ES-1 cells as determined using a clonogenic assay (top panels and graph on left) and a soft agar assay of anchorage independent growth (bottom panels and graph on right). Representative images of individual wells from one experiment are shown. Quantifications represent the mean and standard error of the mean (SEM) of colony counts from independent experiments, each performed in triplicate (colony numbers in controls are set to 1; *p<0.05, relative to control, using the student t-test). (C) Levels of pAkt^Ser473 and total Akt were determined using immunoblotting as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116895#pone.0116895.g001" target="_blank">Fig. 1</a>, and quantified by densitometry. Plot shows relative pAkt^Ser473/Akt ratios from multiple experiments in control and PIK3R3-depleted SK-ES-1 cells; to facilitate comparison among different experiments, one value from control cells in each experiment is arbitrarily set to 1; bars show mean and standard deviation for each group. Statistical analyses did not reveal significant differences among the groups.</p

Expression of PTEN and PIK3R3, and degree of Akt phosphorylation, in Ewing Sarcoma cell lines.

<p>The indicated Ewing Sarcoma cell lines (TC71, SK-N-MC, SK-ES-1, EWS502 and A673) were cultured in their respective media, and harvested at similar confluence (~70%) for protein extract preparation. Expression levels of PIK3R3, PTEN, pAkt (Ser473) and (total) Akt were determined using immunoblotting, with tubulin as the loading control; PIK3R3/tubulin ratios were determined by densitometry, with the ratio in TC71 cells arbitrarily set to 1.</p

PTEN expression in patient tumor samples.

<p>PTEN immunohistochemical staining in a representative PTEN-positive tumor and a PTEN-negative tumor. H+E histology and CD99 immunohistochemical staining are also shown. White arrowheads indicate representative vascular endothelial cells (strongly PTEN+ and CD99-); black arrowheads indicate representative tumor cells (CD99+). Note that vascular endothelial cells in both tumors show strong PTEN immunoreactivity of similar intensity (white arrowheads), but tumor cells only in the top tumor are PTEN immunoreactive (black arrowheads).</p

Re-expression of PTEN in PTEN-negative Ewing Sarcoma cell lines.

<p>PTEN re-expression was achieved using a stable lentiviral expression system, and verified by immunoblotting with tubulin as loading control (C: control cells stably transduced with empty expression vector; P: cells stably transduced with PTEN expression construct; for reference, levels of endogenous PTEN in A673 cells are shown in the same blot). Consequences of PTEN re-expression on Akt phosphorylation (Ser473) are shown below, with quantification of pAkt^Ser473/Akt ratios from independent experiments shown on right (mean and standard deviation; values in control cells set to 1; *p<0.05, relative to control, using the student t-test).</p

PTEN expression and response of Ewing Sarcoma cells to conventional chemotherapeutic agents.

<p>(A) Cell survival upon treatment with the indicated agents was compared between PTEN-positive (PTEN+) and PTEN-negative (PTEN-) Ewing Sarcoma cell lines using an MTT assay (IC50 analysis based on data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116895#pone.0116895.s001" target="_blank">S1 Fig.</a>). IC50 values were determined using a non-linear regression plot. Data represent the mean and standard error of the mean of independent experiments, each performed in triplicate; *p<0.05, using the student t-test. (B) Relative survival of control (dashed lines) and PTEN-re-expressing (solid lines) cells in response to vincristine was determined using an MTT assay. Results represent the mean and standard error of the mean of independent experiments, each performed in triplicate; *p<0.05, using the student t-test.</p

Effects of PTEN re-expression on the response to IGF-1R blockade in PTEN-negative Ewing Sarcoma cell lines.

<p>Relative survival of control (dashed lines) and PTEN re-expressing (solid lines) cells in response to the IGF-1R small molecule inhibitor OSI-906 was determined using an MTT assay, as described in Methods. Results represent the mean and standard error of the mean of independent experiments, each performed in triplicate; *p<0.05, using the student t-test.</p