BCL11B Is Up-Regulated by EWS/FLI and Contributes to the Transformed Phenotype in Ewing Sarcoma

<div><p>The EWS/FLI translocation product is the causative oncogene in Ewing sarcoma and acts as an aberrant transcription factor. EWS/FLI dysregulates gene expression during tumorigenesis by abnormally activating or repressing genes. The expression levels of thousands of genes are affected in Ewing sarcoma, however, it is unknown which of these genes contribute to the transformed phenotype. Here we characterize BCL11B as an up-regulated EWS/FLI target that is necessary for the maintenance of transformation in patient derived Ewing sarcoma cells lines. BCL11B, a zinc finger transcription factor, acts as a transcriptional repressor in Ewing’s sarcoma and contributes to the EWS/FLI repressed gene signature. BCL11B repressive activity is mediated by the NuRD co-repressor complex. We further demonstrate that re-expression of SPRY1, a repressed target of BCL11B, limits the transformation capacity of Ewing sarcoma cells. These data define a new pathway downstream of EWS/FLI required for oncogenic maintenance in Ewing sarcoma.</p> </div

Re-expression of SPRY1 limits the transformation potential of Ewing sarcoma cells.

<p>A. Western blot shows expression of 3xFLAG SPRY1 construct in A673 cells. B. Growth rates of A673 cells expressing 3xFLAG SPRY1 were determined using a 3T5 assay. P-values were determined using a Student’s T-test (ns for not significant).C. Anchorage independent growth of A673 cells expressing 3xFLAG SPRY1 was assessed by the ability to grow in methylcellulose. Error bars represent SD of two technical repeats. P-values were determined using a Student’s T-test (* for p≤0.05). D. Western blot shows levels of phosphorylated and total ERK1/2 when A673 cells are grown under adherent or suspension conditions in the presence or absence of 3xFLAG SPRY1 cDNA. Tubulin is used as a loading control and flag shows 3xFLAG SPRY1 expression.</p

BCL11B is necessary for the maintenance of transformation in Ewing sarcoma cells.

<p>A. A673 and TC71 Ewing sarcoma cells were infected with retroviral shRNA constructs targeting BCL11B (BCL11B-4 and BCL11B-6 shRNA), or luciferase (Luc) as a control. BCL11B levels were determined by western blot. Tubulin was used as a loading control. B. Growth rates of A673 and TC71 cells harboring the indicated shRNA retroviral constructs were determined using a 3T5 assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059369#pone.0059369-Lessnick1" target="_blank">[34]</a>. P-values were determined using a Student’s T-test comparing all conditions to control (Luc shRNA) (* for p≤0.05). C. Anchorage independent growth of control (Luc shRNA) and BCL11B (BCL11B-4 and BCL11B-6 shRNA) knock-down A673 and TC71 cells was assessed by the ability to form colonies in methylcellulose. Error bars represent SD of two technical replicates. P-values were determined using a Student’s T-test comparing all conditions to control (Luc shRNA) (* for p≤0.05, *** for p≤0.001).</p

BCL11B mediates transcriptional repression via the NuRD complex.

<p>A. shRNA knock-down of CHD4 in A673 cells results in the up-regulation of BCL11B repressed genes as measured by qRT-PCR. B. A673 cells treated with the indicated dose of vorinostat for 48 hours results in the dose-dependent increase of the indicated BCL11B repressed genes as measured by qRT-PCR. C. qRT-PCR of A673 cells treated with the indicated dose of the LSD1 inhibitor, HCI-2509, for 48 hours. Error bars represent SD of three technical replicates. P-values were determined using a Student’s T-test comparing all conditions to control (Luc shRNA (A) or DMSO (B,C)) (* for p≤0.05, ** for p≤0.01, *** for p≤0.001).</p