MiR-193b negatively regulates its conserved, predicted binding site in the ErbB4 proximal 3’UTR.

<p>(A) Schematic of the ErbB4 3’UTR with location of predicted conserved miR targeting sequences; wild-type and mutant miR-193b target sequences used in reporter assays (the mutated nucleotides are underlined). (B) A673 and SK-ES-1 cells were transfected with a psiCHECK-2 reporter construct containing the wild-type ErbB4 3’UTR miR-193b site or the mutated site, and either mock transfected, or cotransfected with miR-193b mimic or non-targeting negative control mimic. Luciferase reporter activity, normalized to Renilla luciferase activity, was quantified by luminometry; Luciferase/Renilla activity ratio was arbitrarily set to 1 in the mock-transfected group; mean and standard deviation of triplicate transfections.</p

Functional miniscreen for growth suppressive microRNAs in Ewing Sarcoma.

<p>(A) Ewing sarcoma A673 and SK-ES-1 cells were mock transfected, or transfected with the indicated miR or non-targeting negative control mimics at a final concentration of 25 nM. 24 hours later, cells were harvested, counted and plated at 5 x 10³ cells per well in 96-well plates. Relative cell numbers on day 5, normalized to the mock group and plotted as the mean and standard error of the mean (SEM) of 4 independent experiments, each performed in triplicate, are shown. (B) Ewing sarcoma A673 and SK-ES-1 cells were transfected as in “A” and 24 hours later were plated at 500 cells per well in 6-well plates. Colonies were stained with crystal violet 14 days later, and quantified using a Nikon digital image analysis system (NIS-Elements). Results are represented as the mean and SEM of 4 independent experiments, each performed in triplicate, normalized to the mock group. *: p<0.05, in both A673 and SK-ES-1 cells, relative to negative control (“Neg”, which is the average of two different non-targeting negative control mimics).</p

MiR-193b expression.

<p>Relative miR-193b levels in human mesenchymal stem cells (hMSC) and four different Ewing Sarcoma cell lines (A673, SK-ES-1, TC71 and EWS502), as measured using RT-qPCR with U6 as the internal control; mean and standard deviation of triplicate samples, normalized to hMSCs.</p

MiR-193b negatively regulates ErbB4 protein expression in Ewing Sarcoma.

<p>Ewing sarcoma A673 and SK-ES-1 cells stably expressing empty vector control or miR-193b were harvested for protein extract preparation, and immunoblotting with antibody to ErbB4 and tubulin as loading control.</p

Stable ectopic expression of miR-193b inhibits anchorage-independent growth in soft agar.

<p>Ewing sarcoma A673 and SK-ES-1 cells were stably transduced with the pMSCV-Puro retroviral vector expressing miR-193b, or empty vector (EV) control. (A) Relative miR-193b expression levels following selection were determined using RT-qPCR, with U6 as the internal control; mean and standard error of the mean (SEM) of two independent experiments, each performed in triplicate, normalized to empty vector control. (B) For soft agar assays, cells were harvested, counted and plated at 10,000 cells per well in 6-well plates containing 0.4% agar and growth medium with 20% serum. Colonies were stained with nitroblue tetrazolium 2–3 weeks later, and quantified using Nikon digital image analysis system (NIS-Elements). Results are represented as the mean and standard error of the mean (SEM) of 3 independent experiments, each performed in triplicate (*: p<0.05, relative to empty vector control); images from one experiment are also shown.</p

Depletion of ErbB4 results in inhibition of anchorage-independent growth in soft agar.

<p>ErbB4 was stably depleted using 2 different shRNAs in A673 and SK-ES-1 cells, as confimed by immunoblotting with ErbB4 antibody and tubulin as loading control. Compared to scrambled shRNA control, ErbB4 knock-down resulted in diminished colony formation with both shRNAs, in both cell lines. Graphs show mean and standard error of the mean of two to three independent experiments, each performed in triplicate; colony counts are normalized to the control, which is arbitrarily set to 1; *p<0.05 using the student t-test. Images from one representative experiment are also shown.</p

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

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

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 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