BMI-1 promotes ewing sarcoma tumorigenicity independent of CDKN2A repression

Abstract

Deregulation of the polycomb group gene BMI-1 is implicated in the pathogenesis of many human cancers. In this study, we have investigated if the Ewing sarcoma family of tumors (ESFT) expresses BMI-1 and whether it functions as an oncogene in this highly aggressive group of bone and soft tissue tumors. Our data show that BMI-1 is highly expressed by ESFT cells and that, although it does not significantly affect proliferation or survival, BMI-1 actively promotes anchorage-independent growth in vitro and tumorigenicity in vivo. Moreover, we find that BMI-1 promotes the tumorigenicity of both p16 wild-type and p16-null cell lines, demonstrating that the mechanism of BMI-1 oncogenic function in ESFT is, at least in part, independent of CDKN2A repression. Expression profiling studies of ESFT cells following BMI-1 knockdown reveal that BMI-1 regulates the expression of hundreds of downstream target genes including, in particular, genes involved in both differentiation and development as well as cell-cell and cell-matrix adhesion. Gain and loss of function assays confirm that BMI-1 represses the expression of the adhesion-associated basement membrane protein nidogen 1. In addition, although BMI-1 promotes ESFT adhesion, nidogen 1 inhibits cellular adhesion in vitro. Together, these data support a pivotal role for BMI-1 ESFT pathogenesis and suggest that its oncogenic function in these tumors is in part mediated through modulation of adhesion pathways.

Figures

Figure 1. Ewing Tumors (ESFT) express BMI-1

A.) Gene expression profiling using Affymetrix U133A GeneChips™ detects expression of BMI-1 (Pcgf-4; Probeset ID 202265_at) in 94 primary ESFT and 10 ESFT cell lines. The dashed lines and error bars represent median expression levels and inter-quantile ranges, respectively. B.) Quantitative RT-PCR of 20 primary ESFT and 6 ESFT cell lines confirms detectable but variable expression of BMI-1 in ESFT. Expression in non-malignant H9 human embryonic stem cells (hESC), four primary human bone marrow-derived mesenchymal stromal cell cultures (MSC1-MSC4), and MRC5 lung embryo fibroblasts, is uniformly low. Expression levels were repeated in triplicate and normalized relative to the median expression of GAPDH and ACTIN in the same sample. C.) Western blot analysis detects BMI-1 protein in ESFT cell lines while expression in normal fibroblasts (MRC5) and MSC is very low to undetectable. The variability of protein expression in both ESFT and non-malignant cell lines correlates with transcript expression shown in (B). D.) Immunohistochemical analysis of 67 ESFT biopsy samples reveals that in 49 cases tumor cells are highly positive for BMI-1 (e.g. Case I and II). Unlike tumor cells, endothelial cells in Case I (arrow) and infiltrating lymphocytes in Case II (circled) are negative. In 18 cases (e.g. Case III), BMI-1 is weakly expressed or is only expressed by rare cells.

Figure 2. BMI-1 knockdown does not affect ESFT cell proliferation or death

A.) Quantitative RT-PCR confirms knockdown of BMI-1 following transfection of siRNA oligonucleotides (siBMI1A & siBMI1B) compared to mock or control (siNS) transfected cells. ESFT cell lines: A4573, StaET8.2, TC71 and MCF7: positive control, breast cancer cell line. RNA was harvested from cells 48 hrs post-transfection and gene expression normalized to GAPDH expression levels in the same sample. Bars represent the average and error bars the standard deviations of 3 replicate experiments. B.) siRNA-mediated knockdown of BMI-1 significantly inhibits growth of MCF7 but not ESFT cells. Cells transfected with siRNA oligonucleotides were counted daily for 4 days and the number of viable cells plotted. BMI-1 knockdown (siBMI1) counts were compared at each time point to control (siNS) transfected cells. The average counts are graphed with error bars representing standard deviations of 3 replicate experiments. Data is shown for siBMI1B. Equivalent results were obtained for siBMI1A sequence (not shown). C.) Quantitative RT-PCR analysis shows no impact of BMI-1 knockdown on CDKN2A expression in serum free culture. Stably transduced A4573 cells (shBMI1 and shNS control) were grown in serum-free conditions for 48 hrs prior to RNA isolation. Histogram represents average and standard deviation (error bars) of replicate experiments with gene expression expressed relative to GAPDH in the same samples. D.) Knockdown of BMI-1 has no effect on ESFT cell proliferation or death in cells grown in serum-free conditions. A4573 cells stably transduced with a BMI-1 hairpin construct (shBMI1) and grown in serum free conditions for 48 show no change in cell death (left panels; FACS analysis of Annexin-V/propidium iodide (PI) stained cells) or cell proliferation (right panel; FACS analysis of fixed, PI stained cells).

Figure 3. BMI-1 promotes the anchorage independent of ESFT cells

A.) Phase contrast images of A4573 cells growing in standard culture conditions. siRNA-mediated knockdown of BMI-1 (siBMI1) causes A4573 cells to flatten while lipofectamine-treated and control transfected (siNS) cells continue to grow as 3-dimensional clusters. B.) Stable knockdown of BMI-1 (shBMI1B) impairs colony formation of ESFT cells in soft agar. Images are of macroscopic colonies on the day at which control cell (shNS) media was depleted for each of the respective 3 cell lines. Each well is representative of 6 replicate wells for each condition. Similar results were obtained with a second BMI-1-targeted shRNA construct (see Supplementary Data Figure 4). C.) ESFT cells transduced to over-express BMI-1 (pBp-BMI1) have a reduced time to macroscopic colony formation and increased colony number in soft agar when compared with control, empty-vector-transduced cells (pBp-V). Images are of macroscopic colonies on the day at which pBp-BMI1 media was depleted for each of the respective 3 cell lines. Each well is representative of 6 replicate wells for each condition.

Figure 4. BMI-1 promotes ESFT tumorigenicity in vivo

A.) NOD-SCID mice injected with TC71 cells that express reduced levels of BMI-1 (shBMI1) show delayed engraftment and slowed in vivo growth compared to mice injected with control (shNS) transduced cells (Two-tailed paired t-test p<0.001). B.) Time to tumor cell engraftment is decreased and tumor growth enhanced in NOD-SCID mice injected with TC71 cells that over-express BMI-1 (pBp-Bmi1) compared with empty vector (pBp-V) transduced cells. (Two-tailed paired t-test p=0.01). C.) Q-RT-PCR analysis of RNA harvested from tumor xenografts confirms differential expression of BMI-1.

Figure 5. BMI-1 accelerates ESFT cell adhesion in vitro

A.) A4573 cells were plated in 96 well plates and monitored for adhesion. As shown, cells with stable knockdown of BMI-1 (shBMI1) demonstrate reduced adhesion relative to control cells (shNS) (upper panel) whereas adhesion is accelerated in BMI-1 over-expressing cells (pBp-BMI1) in comparison to empty vector cells (pBp-V) (bottom panel). Data show the mean absorbance of crystal violet taken up by adherent cells in at least 7 replicate wells from duplicate experiments at 30 minute intervals. B.) Quantitative RT-PCR validates that two cell adhesion genes identified by microarrray analysis are truly BMI-1-responsive. Note that although basal levels of the two genes differ, knockdown of BMI-1 leads to significant up-regulation of NID1 and down-regulation of VEZT in all three ESFT cell lines. C.) Western blot confirms up-regulation of nidogen 1 protein in A4573 cells following BMI-1 knockdown (siBMI1A, siBMI1B) and down-regulation in the presence of BMI-1 over-expression (pBp-BMI1). D.) A4573 cells were transfected with siNID1 or siNS control oligonucleotides and knockdown confirmed after 48 hrs by quantitative RT-PCR (left panel). Transfected cells were plated in 96 well plates and adhesion measured as described in (A). As shown, reduced expression of NID1 accelerates adhesion.