ETS proteins promote expression and regulate function of the ... - Nature

Oncogene (2010) 29, 3134–3145

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ORIGINAL ARTICLE

EWS/ETS proteins promote expression and regulate function of the homeodomain transcription factor BRN3A DM Gascoyne1, J Dunne2, S Behjati1, NJ Sebire3, J Anderson1 and DS Latchman4 1 Molecular Haematology and Cancer Biology Unit, University College London Institute of Child Health, London, UK; 2Cancer Research UK Medical Oncology Laboratory, Barts and the London School of Medicine, London, UK; 3Department of Paediatric Pathology, Great Ormond Street Hospital, London, UK and 4Birkbeck College, University of London, London, UK

Ewing’s sarcoma family tumors (ESFTs or EFTs) express neuronal markers, which indicates they may originate from cells at least partly committed to neuronal lineage. However, recent publications suggest EFT originates in mesenchymal stem cells, and EWS/ETS fusion proteins characteristic of EFT activate neuronal marker expression to confer a neural phenotype on EFT. Here we show that the neuronal marker BRN3A/POU4F1 is expressed abundantly at the protein level in primary EFT but not in rhabdomyosarcoma and neuroblastoma, and EFT cells exhibit high activity of the BRN3A proximal autoregulatory region. EWS/FLI-1 siRNA reduces BRN3A expression and promoter activity and EWS/ETS proteins are bound to the BRN3A locus, suggesting a direct function for EWS/ETS proteins in control of BRN3A expression. Differentiation-associated and autoregulatory activities of BRN3A are respectively impaired and altered in EFT cells, and EWS/FLI-1 siRNA can restore some BRN3A function. A potentially novel function for BRN3A in EFT cells is identified. These results extend the hypothesis that EWS/ETS proteins induce expression of neuronal markers such as BRN3A in EFT by showing that the function of those same markers may be restricted or controlled in an EWS/ETS-dependent manner. Oncogene (2010) 29, 3134–3145; doi:10.1038/onc.2010.72; published online 29 March 2010 Keywords: EWS/ETS; Ewing’s sarcoma; EFT; BRN3A; POU4F1

Introduction Ewing’s family tumors (EFTs), including Ewing’s sarcoma and peripheral neuroectodermal tumor, are poorly differentiated often aggressive malignancies exhibiting frequent metastasis and limited expression of neuronal markers such as neuron-specific enolase, S-100 and synaptophysin (Thiele, 1990; Baird et al., Correspondence: Dr DM Gascoyne, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Level 4 Academic Block, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK. E-mail: [email protected] Received 3 June 2009; revised 12 January 2010; accepted 26 January 2010; published online 29 March 2010

2005). EFTs are characterized at the molecular level by specific chromosomal translocations fusing EWS or FUS with ETS family genes, most frequently the prototypical t(11;22)(q24:q12) generating an EWS/FLI-1 fusion (Delattre et al., 1992; Arvand and Denny, 2001). EWS/ETS proteins are aberrant transcriptional regulators that appear to activate oncogenic signaling pathways through both DNA-binding dependent and independent pathways (May et al., 1993; Jaishankar et al., 1999; Arvand and Denny, 2001; Welford et al., 2001). Reduction of EWS/FLI-1 expression decreases EFT cell growth in vitro and tumor-forming capacity in vivo, showing that these oncoproteins maintain cellular proliferation and survival (Tanaka et al., 1997; Prieur et al., 2004; Hu-Lieskovan et al., 2005a). Accordingly EWS/ETS proteins regulate expression from the proliferation-associated MYC, CCND1, TGFBR2, CDKN1A and p57KIP genes (Bailly et al., 1994; Hahm et al., 1999; Dauphinot et al., 2001; Nakatani et al., 2003; Zhang et al., 2004). EWS/ETS proteins also regulate differentiation of a variety of cell types (Eliazer et al., 2003; Torchia et al., 2003; Rorie et al., 2004; Riggi et al., 2005; Hu-Lieskovan et al., 2005b; Yeny et al., 2005). Recent key studies indicate that ectopic EWS/ FLI-1 expression in primary murine bone-derived cells is sufficient to drive tumorigenesis with an EFT-like neural phenotype (Riggi et al., 2005; Yeny et al., 2005). A number of differentiation-associated EWS/ETS target genes have been validated including FRINGE and ID2 (May et al., 1997; Fukuma et al., 2003). We have shown previously (Gascoyne et al., 2004) that EWS/FLI-1 can interact with and, in neural progenitor-type ND7 cells, regulate function of the POU homeodomain factor Brn3a/Pou4f1, hereafter referred to as Brn3a. In addition, results from a study conducted before the discovery of EWS/ETS translocations in EFT suggested that BRN3A might be expressed in such tumors (Collum et al., 1992), and the BRN3A promoter is active in the Ewing’s sarcoma cell line STA-ET-1 (Frass et al., 2002). In the mouse, Brn3a is expressed in specific post-mitotic neurons in the peripheral and central nervous systems (Fedtsova and Turner, 1995), and loss of many of these neurons in Brn3a-null mice indicates a critical requirement for Brn3a in some neuronal specification and survival (McEvilly et al., 1996; Xiang et al., 1996; Eng et al., 2001). Furthermore, exogenous Brn3a is sufficient to

Regulation of BRN3A in Ewing’s sarcoma DM Gascoyne et al

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induce differentiation phenotypes such as growth arrest, apoptosis resistance and neurite outgrowth in sensory neuronal progenitors (Smith et al., 1997; Ensor et al., 2001; Gascoyne et al., 2004). Brn3a also regulates expression from its own Brn3a/Pou4f1 locus in vivo and in vitro, and binds within the locus at conserved distal and proximal autoregulatory enhancer regions (Trieu et al., 2003). We hypothesized that BRN3A might be a component of the neuronal signature induced by EWS/ETS proteins, and that conversely its differentiation and autoregulatory functions might be perturbed in EFT cells. Here we show that BRN3A is expressed in a directly EWS/ETS-dependent manner in EFT, and that it is unable to activate neuronal differentiation of EFT cells. Strikingly, also BRN3A autoregulation function is aberrant in EFT cells and EWS/ETS proteins contribute to this effect. These data demonstrate both upregulation of expression and functional regulation of the same neuronal marker gene by EWS/ETS proteins. Results BRN3A is expressed in primary EFT and EFT-derived cells EWS/FLI-1 can interact with and regulate specific Brn3a functions when expressed in neuronal cells (Gascoyne et al., 2004). However, EWS/FLI-1 and BRN3A must be coexpressed for this interaction to be significant in vivo. To study whether BRN3A protein is expressed in EWS/ETS-positive tumors, we subjected a panel of small round cell tumor (SRCT) types to immunostaining with Brn3a antiserum. Results showed that BRN3A is present in most EFT tumors analyzed (Figure 1a and Table 1). The intensity of staining was similar to that obtained with PC-3 prostate cancer cells overexpressing Brn3a (data not shown), indicative of abundant expression in many tumor cells. EFT samples exhibited strong nuclear staining, with occasional weak cytoplasmic staining in some tumors also being present in IgG controls and thus classified as negative. In contrast, BRN3A was found to be weakly, focally or not expressed in a similar number of non-EFT SRCTs. Positive nuclear staining in EFT samples was validated in a further tissue array containing fragments of seven additional tumors (Figure 1a and Table 1). Immunoblotting for BRN3A was then performed on cell lysates from SRCT cell lines. BRN3A protein was detectable in all EFT lines tested but was found to be absent from RD (rhabdomyosarcoma, RMS), SH-SY5Y and LAN-5 (neuroblastoma, NB) cells (Figures 1b, 3d and data not shown). Some expression was detectable in RH30 (RMS) cells. Importantly, although 32-kDa short and 45-kDa long Brn3a isoforms have been described, EFT cells express predominantly the long isoform of BRN3A. Combined these data indicate that BRN3A protein expression is generally absent from NB cells, present but weak in RMS cells and strongest in EFT cells. Expression in neural progenitor/NB hybrid ND7 cells is likely to derive from their progenitor component. Results

previously describing BRN3A transcript expression in approximately 70% of EFT and derived cell lines but not in NB samples (Collum et al., 1992) correlate well with our findings. We confirmed expression of BRN3A mRNA by realtime PCR (Figure 1c), and these results suggested active BRN3A transcription in EFT cells. Brn3a transcription can be regulated through a conserved upstream proximal autoregulatory element (Trieu et al., 1999, 2003), and to study transcriptional regulation of Brn3a, we constructed a reporter containing this region. Activity from this Brn3a-autoreg reporter was found to be higher in EFT cells than in NB or RMS cells (Figure 1d), suggesting EWS/ETS transcriptional regulators might promote BRN3A expression. To determine whether EWS/ETS proteins contribute to BRN3A transcription in EFT, we reduced EWS/FLI1 expression in RH1 cells by siRNA and monitored BRN3A expression. Significant reduction of EWS/FLI-1 expression was confirmed by real-time PCR (P ¼ 0.011, Figure 1e) and immunoblot (Figure 1f) analyses, whereas increased transcription from the EWS/ETSrepressed TGFbRII promoter (Hahm et al., 1999) validated functional EWS/FLI-1 reduction (Supplementary Figure 1a). Reduction of EWS/FLI-1 significantly decreased both BRN3A transcript levels and Brn3aautoreg activity (P ¼ 0.002 and 0.028 respectively, Figures 1g and h), and BRN3A protein levels (Figures 1f and 4b). Inhibition of Brn3a-dependent neuronal differentiation is a common property of EWS/ETS proteins Individual Ewing’s tumors express different EWS/FLI-1 splice variants (most commonly types I–III) or other fusion proteins such as EWS/ERG and EWS/ETV1 (Arvand and Denny, 2001). BRN3A is expressed in EFT samples containing EWS/FLI-1 type I, II or III proteins (Table 1), and EWS/FLI-1 type I in RH1 cells promotes BRN3A expression (Figures 1f–h). EWS/FLI-1 type IV is sufficient to impair growth arrest and neuronal differentiation initiated by Brn3a (Gascoyne et al., 2004). If inhibition of BRN3A function is important throughout EFT then such regulation should be exhibited by all EWS/ETS fusions. Thus we examined whether, as with EWS/FLI-1, induction of neuronal differentiation by Brn3a can be inhibited by EWS/ERG and EWS/ETV1. Expression of EWS/ETS proteins in transfected ND7 cells was confirmed by immunoprecipitation and immunoblotting (Figure 2a) and functionally by repression of transcription from the TGFbRII reporter (Supplementary Figure 1b). The ability of Brn3a long isoform to significantly activate transcription from a p21WAF1 luciferase reporter (Po0.001, as per Perez-Sanchez et al., 2002) was abolished not only by EWS/FLI-1 but also by EWS/ERG and EWS/ETV1 (P ¼ 0.056, 0.077 and 0.498, respectively, Figure 2b). Importantly, Brn3a-dependent neurite outgrowth was significantly inhibited by all three EWS/ETS fusion proteins (Figure 2c, left-hand panels and Figure 2d, P ¼ 0.044, Oncogene


3136 Screen 1 α-BRN3A EFT

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Figure 1 BRN3A is expressed in primary Ewing’s family tumor (EFT) and EFT cell lines. (a) Primary tumor sections (Screen 1) or tumor fragments (Tissue array) were immunohistochemically stained using rabbit Brn3a antiserum (a-BRN3A) or isotype control (IgG). Positive brown staining was present only in cell nuclei visualized blue by hematoxylin counterstain. The spectrum of staining intensities is shown from strong to moderate positive (EFT) through weak positive (rhabdomyosarcoma, RMS) to negative (neuroblastoma, NB). (b) Small round cell tumor (SRCT) cell lines and ND7 (positive control) were subjected to immunoblot analysis using murine Brn3a antiserum. Equal loading was confirmed by anti-actin re-probing. (c) BRN3A transcript expression in SRCT cells by real-time PCR. (d) Brn3a-autoreg reporter activity in SRCT cells 48 h after transfection. Activities in RH1, SKNMC and RH30 were significantly higher than in SH-SY5Y (Po0.05). (e–h) RH1 were transfected with negative control siRNA (siCON) or siRNA directed against EWS/FLI-1 (siE/F), cultured for 48 h and then either harvested for real-time PCR (e, g) or immunoblot (f) analyses, or passaged for further transfection with Brn3a-autoreg reporter plasmid and luciferase assay (h). Oncogene


3137 Table 1 Number

BRN3A protein expression is higher in EFT than in other SRCT SRCT type

Other details

BRN3A intensity

BRN3A distribution

Tumor presentation

Screen 1 (tumor sections) 1 EFT 2 EFT 3 EFT 4 EFT 5 EFT 6 EFT 7 EFT 8 EFT 9 EFT 10 Rhabdo 11 Rhabdo 12 Rhabdo 13 Rhabdo 14 Rhabdo 15 Neuroblastoma 16 Neuroblastoma 17 Synovial sarcoma

E/F tp I E/F tp III E/F tp II E/F tp I E/F tp II E/F tp I E/F tp I E/F tp I E/F tp I Alveolar P3/F Alveolar P3/F Embryonal Alveolar P3/F Embryonal Poorly diff. Poorly diff. SYT/SSX

þþþ þþþ þþ þþþ þþþ þþþ þþ þ ± ± þ þþ þ þ

Diffuse Diffuse Diffuse Diffuse Focal Diffuse Diffuse Diffuse Diffuse Focal Diffuse Focal Diffuse NA Diffuse

NA Metastatic Stage 4 Stage 4 Nonmetastatic Local extension Local extension Stage 4 NA ND ND ND ND ND ND ND ND

Screen 2 (tissue array) 18 EFT 19 EFT 20 EFT 21 EFT 22 EFT 23 EFT 24 EFT 25 Renal sarcoma

E/F tp I Undetected E/F tp I NA NA NA E/F tp I NA

þþþ þþþ þþ þþþ þ þþ

Diffuse Patchy Diffuse Diffuse Diffuse Focal

Nonmetastatic Local extension Nonmetastatic Nonmetastatic Nonmetastatic Nonmetastatic Local extension ND

Abbreviations: EFT, Ewing’s family tumors; SRCT, small round cell tumor; NA, not available; ND, not determined. In screen 1, sections from 17 SRCTs were a-BRN3A immunostained as described in Figure 1. Eight of nine EFT showed significant BRN3A expression, five displaying high-intensity staining ( þþþ ). Only four of eight non-EFT exhibited BRN3A expression, and staining intensity was generally weaker than EFT sections. Staining was classified ‘diffuse’ if across the entire section, or ‘focal’ if observed in less than approximately 5% of the section. Intensity of a-BRN3A staining was higher than average in EFT samples (P ¼ 0.006) and lower than average in non-EFT samples (P ¼ 0.008). Independent immunostaining of a tissue array containing a further eight tumor fragments confirmed nuclear BRN3A positivity in six of seven additional EFTs, of which the only negative tumor did not have presence of EWS/ETS translocation confirmed.

0.049 and 0.022, respectively). To confirm inhibition of BRN3A function in EFT cells, we measured the ability of ectopic Brn3a to induce neurite outgrowth in RH1 cells. Despite robust ectopic expression (Supplementary Figure 1c), Brn3a did not induce neurite outgrowth even 96 h after transfection (Figure 2c, right-hand panels). Similar experiments show murine Brn3a is competent to promote neurite outgrowth in human SH-SY5Y cells (M Calissano, ICH unpublished data). Thus, the presence of endogenous EWS/ETS correlates with inability of ectopic Brn3a to induce differentiation. Brn3a autoregulation is aberrant in EFT cells One of the most validated Brn3a target genes is the Brn3a/Pou4f1 gene itself, which contains several Brn3a binding sites within proximal and distal enhancer elements (Trieu et al., 1999, 2003). To study in greater detail regulation of Brn3a function in EFT cells, we investigated autoregulation in two EFT cell lines and two non-EFT cell lines using the Brn3a-autoreg reporter. First, we determined that exogenous Brn3a is competent to significantly activate transcription from a reporter containing a single Brn3 consensus binding site (Brn3-consensus) but not from the negative control

pGL3-basic in the sensory neuronal/NB-type cells ND7 and SH-SY5Y, and also in EFT cell types RH1 and SKNMC (all Po0.05, Figure 3a). Thus, ectopic Brn3a can be transcriptionally active in EFT cells. In contrast, although activation of Brn3a-autoreg by Brn3a was significant in ND7 and SH-SY5Y cells (both Po0.05), Brn3a repressed transcription from this reporter in RH1 and SKNMC cells. We hypothesized that addition of a potent transcriptional activation domain to Brn3a might enable it to activate rather than repress the Brn3a autoregulatory element in EFT cells. To test this, we generated AD/ Brn3a, a chimeric Brn3a protein having the potent heterologous nuclear factor-kB activation domain added to its N terminus. As anticipated, AD/Brn3a activated transcription from the Brn3-consensus reporter approximately 10-fold more than wild-type Brn3a both in ND7 and RH1 cells (Figure 3b). However, addition of the nuclear factor-kB activation domain to Brn3a increased Brn3a-autoreg activity in ND7 cells, but not in RH1 cells (Figure 3b). These data suggest a robust alteration of BRN3A autoregulation in EFT cells. Overexpression of Brn3a increased endogenous BRN3A transcript levels in RH30 RMS cells but not in RH1 EFT cells 24 h after transfection (Figure 3c), Oncogene


p21WAF1

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3138 40 30 20 10 0 control E/FLI-1 E/ERG E/ETV1

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Figure 2 Inhibition of Brn3a-dependent neuronal differentiation is a common property of EWS/ETS proteins. (a) EWS/ETS expression in ND7 cells 48 h after transfection with pJ4-EWS/ETS vectors, determined by anti-FLAG immunoprecipitation and immunoblotting. (b) Activity of p21WAF1-luciferase reporter in ND7 cells co-transfected with 0.5 mg reporter and 0.25 mg pJ4 or pJ4Brn3a(L) and 0.25 mg pJ4 or pJ4 EWS/ETS per well in 24-well plates. (c) Left-hand panels, ND7 were co-transfected with eGFP-N1 (Clontech) and pJ4-Brn3a(L) plasmids alongside either pJ4 (control) or pJ4-EWS/ETS plasmids. Final plasmid ratio was 1:3:6, respectively. Induction of neurites was clear in approximately 20% of cells transfected with Brn3a and GFP alone (control), but was reduced to approximately 5% in cells co-expressing exogenous Brn3a and EWS/ETS fusion proteins, (d) shows data from a representative experiment. (c) Right-hand panels, RH1 were transfected with pSG5 (control) or pSG5-Brn3a (Brn3a) alongside eGFP-N1 in the ratio 3:1, cultured in normal media and cell morphology observed up to 96 h later.

supporting an altered endogenous BRN3A autoregulatory response in the EFT cell line, whereas after 48 h endogenous transcript levels had been reduced in both cell types. To definitively confirm altered autoregulation of the endogenous BRN3A gene in EFT cells, we generated a Brn3a expression plasmid that enables discrimination of ectopic from endogenous Brn3a by immunoblot. Exogenous Brn3a N-terminally tagged with three copies of the FLAG-epitope migrates more slowly than endogenous Brn3a when subjected to SDS– polyacrylamide gel electrophoresis; however, its expression also increases the amount of faster-migrating untagged Brn3a in ND7 cells (Figure 3d, left panels). This could be due to induction of endogenous protein or production of untagged protein from the introduced construct. Therefore to prevent the latter, we mutated the first four methionine residues of Brn3a to alanine or leucine, and termed the resulting modified plasmid FL-DBrn3a. Transfection of FL-DBrn3a into BRN3Anegative SH-SY5Y cells confirmed that untagged Brn3a is not produced from this construct (Figure 3d, right panels), so we tested the ability of FL-DBrn3a to Oncogene

regulate endogenous BRN3A protein levels in EFT and non-EFT cell types. Ectopic Brn3a increased endogenous BRN3A levels in a dose-dependent manner in ND7 and RH30 cells (Figures 3e, 5- and 10-fold at lowest dose), but strikingly this positive autoregulation was absent or much reduced (approximately 2-fold at lowest dose) in EFT cell types RH1 and SKNMC. Thus, these data show that BRN3A autoregulation is different in EFT compared with other SRCT cells. EWS/ETS proteins control autoregulation of Brn3a and bind to the BRN3A locus EWS/ETS proteins can control Brn3a differentiation function and therefore might control also Brn3a autoregulation in EFT cells. Co-transfection experiments showed that indeed EWS/ETV1 coexpression in ND7 cells is sufficient to inhibit positive autoregulation of the endogenous Brn3a gene by FL-DBrn3a (Figure 4a). Conversely, EWS/FLI-1 siRNA treatment significantly increased the ability of FL-DBrn3a to positively autoregulate the endogenous BRN3A gene


Relative change in normalised luciferase activity induced by Brn3a

3139 Brn3-consensus

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SKNMC FL-ΔBrn3a Brn3a/BRN3A

47 β-Actin

Figure 3 Brn3a autoregulation is aberrant in Ewing’s family tumor (EFT) cells. (a) small round cell tumor (SRCT) cells were transiently transfected with equal amounts of luciferase reporter and either pJ4-Brn3a or empty vector pJ4. Fold change in luciferase activity induced by exogenous Brn3a was calculated and was significantly different to 1 (Po0.05) in all Brn3a-autoreg and Brn3consensus transfections. (b) Activities of Brn3-consensus and Brn3a-autoreg reporters in ND7 and RH1 48 h after transfection under identical conditions with reporter alongside equal amount of pJ4 (control), pJ4-Brn3a (Brn3a) or pJ4-AD/Brn3a (AD/Brn3a) expression vector. (c) Real-time PCR analysis of human-specific endogenous BRN3A expression in RH30 and RH1 cells at the indicated time points after transfection with pSG5 or pSG5-Brn3a vectors. (d) Immunoblot analysis of ND7 (left-hand panels) transfected with pSG5 () or pSG5-FL-Brn3a (3a) and SH-SY5Y (right-hand panels) transfected with 0 (), 1.5 (D3a left) or 3 (D3a right) mg pSG5-FL-DBrn3a, harvested 48 h after transfection. (e) Immunoblot analysis of ND7, RH30, RH1 and SKNMC transfected to approximately equivalent efficiency (50–80%) with pSG5 () or increasing amounts of pSG5-FL-DBrn3a and harvested 48 h after transfection. Equivalent loading and transfer was confirmed by a-actin re-probe.

in RH1 cells (mean fold increase 1.83±0.54, P ¼ 0.042, Figures 4b and c). Direct interaction with the conserved BRN3A/Brn3a locus might explain the effects of EWS/ETS proteins on

BRN3A expression and searches revealed the presence of several potential EWS/ETS binding sites (Figure 4d). To study direct EWS/ETS binding, we performed realtime chromatin immunoprecipitation analyses in EFT Oncogene


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cells. Anti-FLI-1 but not control IgG immunoprecipitates from SKNMC cells were significantly enriched for the positive control GLI-1 locus fragment (P ¼ 0.035, Beauchamp et al., 2009) and for one of the potential binding sites in BRN3A (site 1, P ¼ 0.021, Figure 4e), indicating that EWS/FLI-1 can interact directly with this locus in vivo. Reduction of BRN3A expression in EFT cells is associated with altered migration and CD117 expression Although BRN3A is expressed in EFT cells, its established functions in neuronal differentiation and autoregulation appear to be altered or perhaps restricted, and thus we investigated potentially intact function of BRN3A in EFT cells. Brn3a can positively regulate Bcl-2 expression in the presence of EWS/FLI-1; however, Brn3a overexpression did not consistently confer protection against apoptosis to EFT cells (data not shown). As Brn3a expression has been previously correlated with aggressive metastatic phenotype in neuroendocrine lung cancer (LeblondFrancillard et al., 1997), we considered that Brn3a might regulate metastasis of EFT cells. Indeed, in the initial screen the only EFT exhibiting focal rather than diffuse BRN3A expression was also the only sample to be nonmetastatic (Table 1). This correlation was not observed in the later tissue array possibly due to smaller size of tumor fragments. Second, we reduced BRN3A expression in RH1 cells by siRNA oligonucleotide transfection (Figure 5a, P ¼ 0.001 and Figure 5b), and studied their migration abilities. Significant chemoattractant-dependent migration of control-siRNA treated RH1 cells through Matrigel (mean 5.6-fold, P ¼ 0.006) was not observed in BRN3A-siRNA treated RH1 cells (mean 1.5-fold, not significant) (Figure 5c). One of the molecules whose expression has been linked with metastasis in EFT is C-KIT/CD117, specifically SCF treatment and downregulation of CD117 expression has been associated with decreased metastasis in vivo (Landuzzi et al., 2000). We found that CD117 is expressed on the surface of RH1 and other EFT cell lines (Figure 5d), and that reduction in BRN3A expression significantly reduces both surface CD117 expression on RH1 cells (Figure 5e, quantitated in Figure 5f, P ¼ 0.002) and their C-KIT transcript levels (Figure 5g, P ¼ 0.02). In summary, these studies show that high BRN3A transcription in EFT cells is partially and directly dependent on EWS/ETS oncoproteins, and that in this context these same proteins alter BRN3A function.

Discussion The function of EWS/ETS proteins in EFT is becoming clearer as microarray profiling shows that these fusions induce expression of target genes, some of whose normal function is associated with neuronal differentiation (Rorie et al., 2004; Hu-Lieskovan et al., 2005b; Hippenmeyer et al., 2005; Riggi et al., 2005; Oncogene

Yeny et al., 2005). It has been shown recently that the neuronal marker NKX2.2 is a critical EWS/FLI-1 target gene in Ewing’s sarcoma (Smith et al., 2006). Here, we have identified that BRN3A is another neuronal-type marker upregulated by EWS/ETS. In support of BRN3A as a bona fide EWS/ETS target, we note that other groups have shown BRN3A expression to increase 50-fold after EWS/FLI-1 expression in both LAN5 and NGP9A NB cells (Rorie et al., 2004) and to be maintained in post-mitotic neurons after deletion of the Ets gene Pea3 and overexpression of the EWS/ETS protein EWS-ER81 (Hippenmeyer et al., 2005). However, transient ectopic EWS/ETS expression alone appears insufficient to increase Brn3a expression in ND7 cells (Figure 3b) or SH-SY5Y cells (data not shown). These data support the hypothesis that induction of neuronal target gene expression by EWS/ETS requires a permissive cellular environment (discussed in Riggi et al., 2008). Expression of Brn3a in DRG neurons of mice deficient for one Brn3a allele is comparable to expression in those of wild-type animals, and mice transgenic for Brn3a overexpression have reduced endogenous Brn3a expression in sensory neurons (Trieu et al., 2003). In contrast, exogenous Brn3a can positively autoregulate activity of the Brn3a proximal autoregulatory region in reporter assays in CV-1 epithelial cells in vitro (Trieu et al., 1999, 2003). We show that ectopic Brn3a can positively autoregulate the endogenous BRN3A/Brn3a locus in neuronal and RMS cells. Possibly cell-type-specific differences define positive or negative autoregulation by Brn3a, and mutagenesis of Brn3a-autoreg will provide insight into the factors involved. Alternatively, overexpression experiments in vitro may increase Brn3a expression above a threshold required for positive feed-forward autoregulation, whereas in vivo transgenic Brn3a overexpression may not reach this threshold and support negative autoregulation. Brn3a autoregulation is likely to have a function in normal terminal neuronal differentiation, and importantly, in the presence of EWS/ETS proteins and in EFT cells, such autoregulation is altered. This may result from both direct interactions between EWS/ ETS and Brn3a proteins (Gascoyne et al., 2004) and interactions of these proteins with the Brn3a locus (Lanier et al., 2007 and present study). Our findings show that EWS/ETS proteins are also sufficient to prevent Brn3a function in terminal neuronal differentiation. This correlates well with data showing the ability of EWS/ETS to prevent neuronal differentiation (Rorie et al., 2004; Hippenmeyer et al., 2005). Furthermore, the results of a very elegant study show that EWS/ETS protein expression is sufficient not only to inhibit terminal differentiation of neurons but conversely also able to promote neuronal differentiation functions in developing neurons (Hippenmeyer et al., 2005). Therefore, it appears that EWS/ETS proteins propel expression of neuronal markers and stimulate early stages of neuronal development but prevent terminal neuronal differentiation. We show for the first time that EWS/ETS proteins may perform these


3141 FL-ΔBrn3a EWS/ETV1

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ctgtgctTTCCggcctg

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Figure 4 EWS/ETS proteins bind to and control autoregulation of BRN3A. (a) Immunoblot analysis of ND7 48 h after transfection with pSG5-FL-DBrn3a ( þ ) or pSG5 vector () in the presence of pMSCV () or pMSCV-EWS/ETV1 ( þ ) co-transfection, as indicated. (b, c) Immunoblot analysis of RH1 transfected with EWS/FLI-1 (siE/F, þ ) or control () siRNA, and transfected again after 48 h with both repeat siRNA and pSG5-FL-DBrn3a plasmid or control pSG5 plasmid. Cells harvested after further 48 h. Panel (c) shows quantitation of three independent experiments such as that shown in (b). Fold change induced by FL-DBrn3a is indicated above data bars. (d) Schematic diagram to show potential EWS/ETS binding sites in the BRN3A locus, not to scale. Number indicates base position with respect to the start of Exon 1. Arrows indicate approximate positions of real-time PCR primers used for chromatin immunoprecipitation (ChIP) analysis. (e) Real-time PCR ChIP analysis of EWS/FLI-1 binding to endogenous BRN3A, b-2M (negative control) and GLI-1 (positive control) fragments in SKNMC cells using whole rabbit IgG or specific a-FLI-1 antibodies and amplification oligonucleotides as described in the Materials and methods section.

functions by controlling function of a pro-neuronal protein whose expression they have induced. As Brn3a may function on some target genes as a transcriptional repressor (Trieu et al., 2003), it is possible that aberrant recruitment of the EWS activation domain to such loci through direct protein–protein interaction with BRN3A counteracts such repression. Indeed diverse EWS/ETS proteins sharing a common EWS activation domain inhibit Brn3a function similarly. However, intact function of Brn3a on a consensus response element in EFT cells indicates that EWS/ETS do not interfere with Brn3a DNA binding or cofactor recruitment per se. Therefore, the function of Brn3a at its differentiation-associated target promoters may be impaired in EFT cells through adjacent DNA-dependent binding and possible interaction with aberrant EWS/ETS proteins. Adjacent binding sites for POU proteins and ETS proteins have been reported previously (Bradford et al., 1997), and it may be interesting

to determine whether POU and EWS/ETS proteins function co-operatively at multiple adjacent promoter sites in a manner similar to AP-1 and EWS/ETS complexes (Kim et al., 2006). Direct recruitment of the EWS N-terminal domain to POU binding sites by gene fusion may provide an alternative mechanism for this effect (Yamaguchi et al., 2005). The long Brn3a isoform can be oncogenic when coexpressed alongside Ha-Ras in rat embryo fibroblasts (Theil et al., 1993), and this is the predominant isoform in EFT cells. BRN3A has been proposed to have an intact oncogenic function in EFT (Collum et al., 1992; Theil et al., 1993), and a potential positive correlation of BRN3A distribution in primary EFT samples with invasion (Table 1) provides evidence to support this hypothesis. A similar correlation has been observed in small cell lung carcinoma (LeblondFrancillard et al., 1997). The low frequency of Ras mutations in EFT (Radig et al., 1998) suggests that other oncogenic events Oncogene


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Figure 5 BRN3A regulates expression of CD117 in RH1 cells. (a) Real-time PCR analyses of RH1 cells 48 h after transfection with siCON or siBRN3A oligonucleotides. (b) Immunoblot analysis of cells targeted as in a, 48 h after transfection. (c) Cells targeted as in a were plated into Matrigel-containing chambers in reduced serum medium, above either the same medium (serum starved) or medium containing 10% fetal calf serum and SCF (100 ng/ml). Migration was assessed by MTS activity (absorbance at 490 nm as per manufacturer’s protocol—Promega) within lower wells after a further 48 h. (d) Flow cytometric analyses of exponentially growing EFT cell lines stained with isotype control (dashed line) or anti-CD117 antiserum (gray). (e) Flow cytometric analyses of CD117 expression in RH1 cells targeted as in a with siCON (gray) or siBRN3A (black line). (f) Mean fluorescence intensity of CD117 staining representing duplicates from three independent experiments. (g) Real-time PCR analysis of C-KIT expression in RH1 cells 48 h after targeting as in a, mean of three experiments.

(such as EWS/ETS translocation) may be the events cooperating with BRN3A upregulation in this disease. Our initial studies show that BRN3A can regulate expression of the c-KIT growth factor tyrosine kinase receptor in EFT cells, where such expression (Landuzzi et al., 2000; Gonzalez et al., 2004) has been linked to metastatic potential. C-KIT expression changes may be direct or perhaps due to altered receptor activity, and BRN3A may regulate activity of other tyrosine kinase receptor signaling pathways such as that of the IGF-1R whose important function in EFT has been recently established (Manara et al., 2007). In conclusion, although the neuronal-type marker BRN3A is an EWS/ETS target expressed in EFT, one of the functions of EWS/ETS in regulating neuronal differentiation may lie in their ability to control selective BRN3A transcriptional function. There is a need to improve on current cytotoxic therapies for this disease, but these tumors and cells derived from them are generally refractory to standard differentiation therapies. Thus, restoration of normal BRN3A function or that of other neuronal-type markers may prove therapeutically useful in EFT.

Materials and methods Mammalian cell culture EWS/FLI-1 type I RH1 cells and RMS RD and RH30 cells were cultured in Dulbecco’s modified Eagle’s medium Oncogene

supplemented with 10% fetal calf serum (Sigma-Aldrich, Gillingham, UK). TC-32 (E/F type I), SKNMC and RDES (E/F type II) and NB SH-SY5Y, IMR32 and LAN5 cells were cultured according to American Type Culture Collection recommendations. ND7, a sensory neuronal cell type immortalized by NB fusion (Wood et al., 1990), was cultured at low passage. Plasmid generation The Brn3a-autoreg reporter containing the murine proximal Brn3a autoregulatory region was generated by insertion of a 154-bp PCR product from murine gDNA using oligonucleotides 3aprox.F (50 -AGTTGCCTTGTCATGTAACAC-30 ) and 3aprox.R (50 -CGTTGCACACAATCCTTTAAC-30 ) into pGL3-basic (Promega, Southampton UK) between SacI and XhoI. The reporter Brn3-consensus containing a single Brn3 binding site was generated by cloning complimentary oligonucleotides consensus.F (50 -CTCCTGCATAATTAATTACG CCCG-30 ) and consensus.R (50 -CTAGCGGGCGTAATTAA TTATGCAGGAGGTAC-30 ) into pGL3-promoter (Promega) between KpnI and NheI. The expression plasmid pJ4-AD/ Brn3a coding for the nuclear factor-kB activation domain fused to Brn3a at its N terminus was generated by insertion of Brn3a(L) cDNA from pBluescript-3aL (Gascoyne et al., 2004) into pCMV-AD (Stratagene, LaJolla, CA, USA) using EcoRI and NotI, and subsequent transfer of the AD/Brn3a cassette into a modified pJ4 vector between XbaI and BamHI. To generate pSG5-FL-DBrn3a, we substituted the first four methionine codons of Brn3a(L) cDNA within pBluescript3aL with alanine or leucine codons by replacement of an EcoRI/NsiI N-terminal cDNA fragment with complimentary oligonucleotides 3astartdel.F (50 -AATTCGCGGCTTCCGCA


3143 AACAGCAAGCAGCCTCACTTTGCCTTGCA-30 ) and 3astartdel.R (50 -AGGCAAAGTGAGGCTGCTTGCTGTTTGC GGAAGCCGCG-30 ). The resulting DBrn3a cDNA replaced wild-type Brn3a in pSG5-FL-3aL (Gascoyne et al., 2004). N-terminally FLAG-tagged pJ4-EWS/ETS expression plasmids were cloned from previously described pBluescript plasmids (Gascoyne et al., 2004). pMSCV-EWS/ETV1 was generated by insertion of an EcoRI/BglII fragment containing the FLAG-tagged EWS/ETV1 cDNA from pJ4-FL-EWS/ETV1 into pMSCV-neo (Clontech, Mountain View, CA, USA). Transient transfection For reporter studies, we plated all cell types at 4 104 cells per well in 24-well plates 24 h before transfection with 0.25 mg of reporter plasmid and 0.25 mg of each expression plasmid by calcium phosphate method using 40 ml CaP complex. pRL-TK (10 ng; Promega) was co-transfected to enable normalization for transfection efficiency. Luciferase activities were measured by Dual Luciferase assay (Promega). For expression studies, we plated all cell types in six-well dishes 24 h before transfection. SH-SY5Y at 8 105 cells per well were transfected with up to 3 mg DNA using 8 ml Lipofectamine 2000 (Invitrogen, Paisley, UK). ND7 at 4 105 cells per well were transfected with up to 2 mg DNA by calcium phosphate method using 800 ml of complex. RH1, SKNMC and RH30 plated at 5 105 cells per well were transfected with up to 0.5 mg DNA using 5 ml Lipofectamine (Invitrogen). All cells were harvested at 48 h. For neurite outgrowth, we plated ND7 and RH1 at 2 105 cells per well, transfected, and visualized GFP þ cells by fluorescence microscopy. Images were converted to gray scale and contrast inverted to observe neurites. Immunohistochemistry Initially, representative SRCTs were selected at random from our institution’s archive on the basis of confirmatory molecular diagnosis in accordance with review board approval. A second tissue-array screen comprising fragments from seven further EFTs and a renal sarcoma was stained independently to validate findings. Sections (4 mm) from archival routine formalin-fixed paraffin embedded samples were cut onto slides and heated overnight. After de-waxing and re-hydration, we blocked sections in PBS containing 20% goat serum (Sigma) and 0.1% Triton X-100 (Sigma). Sections were then incubated with Brn3a antiserum (Fedtsova and Turner, 1995) diluted 1:200 in PBS containing 10% goat serum, 0.1% Triton X-100. After PBS washes, we revealed antibody binding by standard techniques (ABC kit; Dako, Ely, Cambs, UK). After dehydration, we counterstained slides with Mayer’s hematoxylin. Control experiments showed that this antibody recognizes a nuclear antigen abundant in extensively formalin-fixed paraffin-embedded PC-3 prostate cancer cells engineered to overexpress Brn3a but exhibits little reactivity against vector-transfected PC-3 cells (data not shown).

Immunoblotting Immunoblotting and immunoprecipitation was performed using standard protocols, as described previously (Gascoyne et al., 2004). Each whole-cell lysate (40 mg) was analyzed, antibodies recognizing Brn3a (MAB1585; Chemicon/Millipore, Watford, UK), FLAG epitope (F-7425; Sigma), FLI-1 (C-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and b-actin (sc-1616; Santa Cruz Biotechnology) being used at 1:1000 dilution.

siRNA transfection RH1 were plated at 1 106 per well in six-well dishes 24 h before transfection using 8 ml Lipofectamine 2000 (Invitrogen). Double-stranded RNA oligonucleotides (MWG Biotech, Ebersberg, Germany) are the following: negative control (siCON) sense strand 50 -GCAAGCUGACCCUGAAGUUC AU-30 , BRN3A (siBRN3A, Hs_POU4F1_2_HP; Qiagen, Crawley, UK), and EWS/Fli-1 type I (siEF) sense strand 50 -GCUACGGGCAGCAGAACCCTT-30 . Migration assay of transfected cells in Matrigel Invasion chambers was performed as per manufacturer’s instructions (BD Biosciences, Oxford, UK). Flow cytometric analysis 48 h after transfection was performed on a CyAnADP (DakoCytomation) after staining cells with isotype or specific anti-human CD117-PE 555714 antibodies (BD). Chromatin immunoprecipitation Briefly, four subconfluent 10 cm plates of SKNMC cells were formaldehyde cross-linked, lysed, sonicated and immunoprecipitated using either a-FLI-1 antibody (C-19; Santa Cruz Biotechnology) or rabbit IgG control. Cross-links in purified complexes were reversed, DNA purified and subjected to realtime PCR analysis alongside input samples using oligonucleotide pairs BRN3A site 1 (50 -GGGCGTGGGAGACGAAA-30 and 50 -TCCCCAGCTGCGAGTTACAT-30 ), BRN3A site 2 (50 -GATGAGCAGGACGTCGCTAGA-30 and 50 -CGGACT CCCGGTGTGATG-30 ), BRN3A site 3 (50 -GGCAGAGGAG GTTCTCAAGGA-30 and 50 -AATTAAACGACCAGACGA AAGAAAAC-30 ), and GLI-1 (50 -CCGGCCGCTGCAAGT-30 and 50 -CTAACTGAGCATTCTGCCATCCT-30 ). Real-time PCR Total RNA was isolated from cell lines using RNeasy columns (Qiagen), and cDNA synthesized (M-MLV Reverse Transcriptase RnaseH from Promega). Universal master mix (Applied Biosystems, Warrington, UK) was used, and normalized transcript expression values calculated by the 2ddC method as per Applied Biosystems user bulletin #2, based on cycle threshold (CT) values and those of the housekeeping gene 18S. Assays were Hs100366711_ml for human POU4F1, Hs00174029_m1 for C-KIT and TaqMan assay 4310893E for 18S (both Applied Biosystems). Oligonucleotides EFampF (50 -TACAGCCAAGCTCCAAGTC-30 ) and EFampR (50 -TTT TGAACTCCCCGTTGGTC-30 ), and SYBRgreen mastermix (Applied Biosystems) were used to amplify EWS/FLI-1 transcripts. Human-specific BRN3A amplification was performed using SYBRgreen and the oligonucleotides 50 -GGAGCCATA ATCTGCAACTTCATT-30 and 50 -AACTTCAACTTCTCGC TCGTTTG-30 . T

Statistical analyses Transfection and chromatin immunoprecipitation data represent mean values from at least three independent experiments, triplicates within each experiment. Neurite outgrowth experiments were performed twice, with each condition in triplicate. Error bars represent standard deviation of means. P-values were obtained using Student’s t-test with unequal variance assumed.

Conflict of interest The authors declare no conflict of interest. Oncogene


3144 Acknowledgements We thank Sue Burchill (Leeds, UK) and Peter Houghton (Memphis, USA) for cell lines, Eric Turner (San Diego, USA) for a-Brn3a antiserum, Seong-Jin Kim (Bethesda,

USA) for TGFbRII (1670/ þ 36), Sian Gibson and Dyanne Rampling for technical assistance and James Diss for PC-3 cells and useful discussions. Funding was provided by MRC and BBSRC (DMG and DSL), and CR-UK (JD).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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