Original Article

Oncogene (2011) 30, 2044–2056; doi:10.1038/onc.2010.582; published online 17 January 2011

Genome-wide screen reveals WNT11, a non-canonical WNT gene, as a direct target of ETS transcription factor ERG

L H Mochmann1, J Bock1, J Ortiz-Tánchez1, C Schlee1, A Bohne1, K Neumann2, W K Hofmann3, E Thiel1 and C D Baldus1

  1. 1Department of Hematology and Oncology, Charité, Campus Benjamin Franklin, Berlin, Germany
  2. 2Institute for Biometrics and Clinical Epidemiology, Charité, Campus Mitte, Berlin, Germany
  3. 3Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany

Correspondence: Dr CD Baldus, Department of Hematology and Oncology, Charité, Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany. E-mail: claudia.baldus@charite.de

Received 14 July 2010; Revised 2 November 2010; Accepted 23 November 2010; Published online 17 January 2011.

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Abstract

E26 transforming sequence-related gene (ERG) is a transcription factor involved in normal hematopoiesis and is dysregulated in leukemia. ERG mRNA overexpression was associated with poor prognosis in a subset of patients with T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Herein, a genome-wide screen of ERG target genes was conducted by chromatin immunoprecipitation-on-chip (ChIP-chip) in Jurkat cells. In this screen, 342 significant annotated genes were derived from this global approach. Notably, ERG-enriched targets included WNT signaling genes: WNT11, WNT2, WNT9A, CCND1 and FZD7. Furthermore, chromatin immunoprecipitation (ChIP) of normal and primary leukemia bone marrow material also confirmed WNT11 as a target of ERG in six of seven patient samples. A larger sampling of patient diagnostic material revealed that ERG and WNT11 mRNA were co-expressed in 80% of AML (n=30) and 40% in T-ALL (n=30) bone marrow samples. Small interfering RNA (siRNA)-mediated knockdown of ERG confirmed downregulation of WNT11 transcripts. Conversely, in a tet-on ERG-inducible assay, WNT11 transcripts were co-stimulated. A WNT pathway agonist, 6-bromoindirubin-3-oxime (BIO), was used to determine the effect of cell growth on the ERG-inducible cells. The addition of BIO resulted in an ERG-dependent proliferative growth advantage over ERG-uninduced cells. Finally, ERG induction prompted morphological transformation whereby round unpolarized K562 cells developed elongated protrusions and became polarized. This morphological transformation could effectively be inhibited with BIO and with siRNA knockdown of WNT11. In conclusion, ERG transcriptional networks in leukemia converge on WNT signaling targets. Specifically, WNT11 emerged as a direct target of ERG. Potent ERG induction promoted morphological transformation through WNT11 signals. The findings in this study unravel new ERG-directed molecular signals that may contribute to the resistance of current therapies in acute leukemia patients with poor prognosis characterized by high ERG mRNA expression.

Keywords:

ETS-related gene (ERG); WNT11; acute leukemia; ERG target genes; 6-bromoindirubin-3-oxime (BIO); morphological transformation

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Introduction

The erythroblastosis virus E26 transforming sequence (ETS) encodes the ETS-related gene (ERG) that has an important physiological role in hematopoiesis (Loughran et al., 2008), angiogenesis (Birdsey et al., 2008), vascular (Ellett et al., 2009) and bone development (Iwamoto et al., 2007). ERG belongs to the highly conserved ETS transcription factor family that is defined by the ETS DNA-binding motif 5′-GGA(A/T)-3′ (Sharrocks, 2001; Wei et al., 2010). In hematopoietic development, ERG has long been postulated to function during early stages of T-cell development as its mRNA abundance peaks and diminishes as cells undergo T-lineage commitment (Anderson et al., 1999). In addition, ectopic ERG expression was shown to induce megakaryocytic differentiation in human K562 cells (Rainis et al., 2005) and in hematopoietic progenitors ERG promotes expansion of megakaryocytes (Stankiewicz and Crispino, 2009). ERG function was further characterized by Loughran et al. in heterozygous mice harboring a missense mutation that phenotypically displayed mild cytopenia in the B-cell compartment and notably had a reduction of progenitor cells by 50%. Furthermore, homozygosity for the same mutation failed to establish definitive hematopoiesis at the embryonic stage. Thus, normal ERG function was necessary to establish and maintain hematopoiesis.

ERG dysregulation has been reported in solid tumors and hematological malignancies. Three fusion proteins composed of ERG with TMPRSS2 (Tomlins et al., 2005; Klezovitch et al., 2008), EWS (Sorensen et al., 1994) or TLS (Kong et al., 1997) create oncogenic proteins. The most frequent chromosomal fusion in prostate cancer consists of the 5′-untranslated region of TMPRSS2 fused with 3′-end ERG. This fusion has multiple variants, of which two of the most common variants are associated with poor outcome (Narod et al., 2008). In leukemia, ERG overexpression is also believed to contribute to the molecular pathogenesis in a subset of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) patients. High ERG expressers were associated with an inferior clinical outcome (Marcucci et al., 2005; Baldus et al., 2006). The pathogenesis of ERG was also observed in sublethally irradiated mice transplanted with ERG transduced progenitor cells, whereby megakaryoblastic leukemia developed (Salek-Ardakani et al., 2009). Thus, several clinical and experimental studies indicate that ERG contributes to the pathogenesis in cancer and leukemia; however, the underlying biological mechanisms are not yet fully understood.

To unravel the molecular function of ERG in acute leukemia, we have conducted a genome-wide screen of ERG target genes. Chromatin immunoprecipitation-on-chip (ChIP-chip) analyses of ERG candidate target genes revealed that ERG may participate in a broader spectrum of biological signaling than previously described. ERG loss and gain of function experiments directly affected WNT11, a non-canonical WNT pathway gene. Moreover, a proliferative growth advantage was observed when ERG-induced cells were treated with WNT agonist 6-bromoindirubin-3-oxime (BIO) and a glycogen synthase-3β (GSK-3β) inhibitor. Finally, co-expression of ERG and WNT11 stimulated morphological transformation of round hematopoietic cells to polarized cells with protrusions upon ERG induction. In addition, the elongation process of ERG-induced cells was effectively inhibited by the addition of BIO and with small interfering RNA (siRNA)-mediated knockdown of WNT11. These findings show that in human leukemia WNT11 is a direct target of ERG and highlight a role for ERG in the WNT signaling pathway.

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Results

Genome-wide screen of ERG candidate genes

ERG transcriptional networks in leukemia are unknown. Thus, in order to construct ERG-related networks, a genome-wide screen by ChIP-chip was conducted in a human T-cell leukemia line, Jurkat. ChIP was performed with two ERG-specific antibodies: single ChIP (C20) and with double (C20 and C17) precipitating antibodies for increased accessibility to multiple ERG epitopes (refer to Materials and methods for a detailed description). Each ChIP with single and double precipitating antibodies was carried out in duplicate. The in vivo assay allowed for the enrichment and identification of ERG-bound DNA sequences that were subsequently hybridized to a high-resolution human promoter chip. Duplicate ChIP-chips 1 and 3 (single-antibody ChIP) resulted in 13070 and 4405 significant peaks, respectively. Duplicate ChIP-chips 2 and 4 (double-antibody ChIP) resulted in 11227 and 6630 significant peaks, respectively. As expected, many significant peaks were detected at individual gene promoters, which yielded 1683, 3066, 1304 and 973 single genes, in ChIP-chips 1–4, respectively. Pooled gene sets from ChIP-chips 1 and 3 are denoted as ChIP-chip I and pooled gene sets from ChIP-chips 2 and 4 are denoted as ChIP-chip II. Finally, only overlapping genes from ChIP-chips I and II were combined to condense the significant candidate gene pool to 342 gene annotations (Table 1). DAVID Functional Annotation Tool (Laboratory of Immunopathogenesis and Bioinformatics, Frederick, MD, USA) was used to characterize biological themes (Table 2). Statistically enriched gene ontology categories suggest a broad functional role for ERG that included developmental processes, multicellular processes, biological adhesion and biological regulation (P-value <0.05). Furthermore, ERG candidate genes subjected to Ingenuity Pathway Analyses revealed an overlap of enriched WNT target genes in several key developmental pathways (Table 3).




Validation of enriched promoter regions and selection of ERG target genes

Based on the gene's described relevance to hematopoiesis and leukemia in the scientific literature (versus a random calculated approach), 24 of the 342 enriched promoter regions were selected for further examination. MATCH algorithm was used to determine the significance and location of conserved ETS-binding motif (5-GGAA/T-3) in each of the 24 putative promoter regions up to 2kb from the transcription start site (TSS) (Table 4 and Supplementary Table 1). In all, 17 of 24 selected targets were confirmed by quantitative PCR, with at least twofold enrichment relative to total chromatin in both experimental ChIPs (Figure 1a). Notably, of these enriched promoter amplicons, genes WNT2, WNT9A, WNT11, CCND1 and FZD7 are participating genes in WNT signaling. Other enriched promoter regions also included hematopoietic genes (CD7, EPO and CD14) and transcriptional regulation genes RBPJL, CDK9, TWIST1, HDAC4, RXRA and OLIG2. Also, TMPRSS2, a component of TMPRSS2ERG fusion oncogene, was significantly enriched in ChIP-chip experiments and was validated by PCR, implicating that ERG may bind the fusion TMPRSS2–ERG promoter to induce expression. In line with previous ERG ChIP analyses, GP1BB, encoding the megakaryocytic CD42c marker, was a significantly enriched promoter region (Bastian et al., 1996).

Figure 1.
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Validation of ERG targets in leukemia. (a) A selection of 24 candidate genes, obtained in duplicate ChIP-chip assays (ChIP-chips I and II), was validated by determining enriched promoter regions via chromatin PCR amplification (ERG-specific immunoprecipitated DNA versus total chromatin) with SYBR Green quantitative PCR. As a control, IgG-immunoprecipitated DNA versus total chromatin was amplified in parallel to ERG-enriched DNA from ChIP-chips I and II and subtracted from fold enrichment values as background. Significant candidate genes with at least twofold enrichment (dotted line) in duplicate ChIPs are shown relative to total chromatin. Each SYBR Green assay was conducted in triplicate wells and values are displayed as averages of three SYBR Green assays. (b) Using SignalMap software from NimbleGen human promoter chip array, fluorescent log2 ratios display multiple peak detection at the chromosomal location of the WNT11 promoter in ChIP-chip 1 and the transcription start site is indicated (TSS) by an arrow.

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A survey of chip hybridization intensity peaks from four hybridizations (ChIP-chips 1–4) for the 17 enriched candidate genes showed several statistically significant peaks mapping to similar genomic coordinates. For example, an overlay of hybridization intensity signals in the WNT11 promoter region for ChIP-chips 1 and 2 showed similar intensity signals spanning similar arrayed genomic regions in Figure 1b (refer to Supplementary Figure 1a for an overlay of hybridizations, ChIP-chips 1–4). The overlay of intensity signals assured ChIP-chip reproducibility of WNT11 promoter enrichment with single and double ChIP precipitating ERG antibodies. Interestingly, WNT11 promoter had the highest number of statistically significant peaks of the 24 putative genes (refer to ranking list, Table 4) and significant peaks clustered at nearly matching genomic coordinates in ChIP-chips 1–4 (Supplementary Figure 1b). In contrast, ERG, and other randomly selected non-target genes, had statistically insignificant or undetectable signal intensities for ChIP-chips 1–4. Thus, genes harboring statistically significant ETS-binding sites in the proximal promoter regions, enrichment of the proximal promoter regions through quantitative PCR and significant multiple peak detection (threshold limit>0.1) in the respective promoter regions collectively determined the 17 candidate genes as putative ERG targets.

ERG occupies the WNT11 promoter in primary T-ALL and AML leukemia blasts

Of the 17 candidate genes, WNT11, in parallel with ERG, has a developmental role in hematopoiesis (Brandon et al., 2000) and was implicated in prostate tumor malignancy (Zhu et al., 2004). Therefore, we focused on WNT11 and examined whether ERG bound to the WNT11 promoter in primary hematopoietic cells. ChIP was conducted with fresh bone marrow cells from one normal donor, one T-ALL patient and five AML patients (AML A, B, C, D and E). WNT11 promoter enrichment was observed in six of seven ChIP bone marrow donors (normal donor, T-ALL, AML C, AML D and AML E) relative to total chromatin (Figure 2). However, a significant linear correlation between ERG mRNA expression and WNT11 promoter enrichment was not observed. We presumed this result was due to the small number of ChIP samples and due to the molecular heterogeneity of acute leukemia. To gain better understanding of ERG and WNT11 relationship, we additionally measured mRNA expression of ERG and WNT11 in diagnostic bone marrow material that included a larger cohort of AML (n=30), T-ALL (n=30) and normal bone marrow (n=30). We observed that ERG mRNA expression was co-expressed with WNT11 mRNA in a considerable fraction (80%) of unselected AML and normal bone marrow samples and to a lower extent of co-expression, 40% in T-ALL bone marrow samples (Supplementary Figure 2a). Despite a nonlinear correlation, WNT11 promoter enrichment through ChIP in primary normal and leukemia cells strengthened that ERG occupied the WNT11 promoter.

Figure 2.
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WNT11 promoter enrichment by ChIP in primary bone marrow cells. ChIP assays with ERG-specific antibody C20 and control IgG in duplicate ChIPs were conducted in primary normal and leukemia bone marrow cells. The bone marrow samples included five AML (AML A–E), one T-ALL and one normal donor. Gray bars display an average of three SYBR Green assays per sample showing fold enrichment of the amplified WNT11 promoter region (the sequence amplified is located from -401 to -501bp relative to TSS) relative to total chromatin. Enrichment of the amplified WNT11 promoter region obtained from ChIP with IgG was subtracted as background from the fold enrichment value. Quantitative PCR was conducted in triplicate wells. For comparison with ERG abundance, the right axis is shown as a plot of relative ERG mRNA expression measured by multiplex quantitative RT–PCR of each of the seven individual bone marrow samples.

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Knockdown of ERG expression results in transcriptional downregulation of ERG target genes

To gain an understanding of relevant targets of ERG in leukemia, siRNA molecules were used to knock down endogenous ERG to determine if mRNA expression of candidate targets was affected. Examination of ERG mRNA expression in Jurkat was quantitatively low compared with other hematopoietic cell lines. Therefore, KG1 cells, having higher ERG mRNA expression, were used to knock down ERG mRNA expression. ERG mRNA expression was efficiently reduced by 70% in KG1 cells. This was further evidenced by western blot, whereby a reduction of 60% (for the 70% ERG knockdown at the transcript level) of ERG protein was observed (Figures 3a and 3b). Furthermore, we determined that 9 of the 17 ERG putative targets were expressed in KG1 cells. Of the nine target genes consistently downregulated by siRNA-mediated ERG knockdown were RASSF1, TWIST1 and RXRA, and those significantly downregulated were TNFRSF25 and WNT11 transcripts (by 40–50% reduction) (Figure 3c). The lack of mRNA expression of several genes in KG1 and in other leukemia cell lines may be due to aberrant gene promoter methylation, as described for WNT2 (Lelinek et al., 2008) and WNT9 (Shu et al., 2006), or requirement of additional factors in leukemia cells.

Figure 3.
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siRNA-mediated knockdown of ERG downregulates gene expression of WNT11 and other ERG putative targets. (a) Multiplex quantitative RT–PCR was conducted to measure relative knockdown of ERG mRNA expression. ERG mRNA knockdown of 70±0.03% is shown. (b) This figure depicts a representative western blot displaying reduced ERG protein levels from siRNA-mediated ERG knockdown in KG1 cells (60% at the transcript level). ERG protein levels of cells transfected with control siRNA luciferase and untransfected KG1 cells remain unchanged. (c) To determine the effects of ERG knockdown on putative target genes, cDNA derived from transfected KG1 cells with ERG-specific siRNA and control siRNA (70% knockdown) were used as templates for the measurement of relative mRNA changes. ERG siRNA-mediated knockdown statistically affected WNT11 and TNFRSF25. Fold changes relative to the control siRNA are displayed as averages of two SYBR Green assays and each assay was conducted in duplicate wells. The asterisk indicates significance by Student's paired t-test (P-value<0.05).

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Induced ERG expression results in transcriptional upregulation of ERG target genes

A controlled ectopic expression of ERG in K562 cells (a cell line normally lacking ERG) was used to examine the cellular consequences of ERG overexpression. A tet-on inducible gene expression system was selected to induce ERG expression by the addition of doxycycline (dox). Stably transfected pTRE-ERG isoform 3 (ERG3) clones in K562 cells were examined for elevated ERG mRNA induction (Figure 4a, black bars) and elevated ERG protein levels (Figure 4b). A potent fold induction of ERG mRNA expression was achieved in individual clones ranging from 10- to 30-fold. These stable clones are referred to hereafter as ERG-inducible cells. In the absence of dox, ERG expression was low or not measurable.

Figure 4.
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ERG induction upregulates gene expression of WNT11 and other ERG putative targets. (a) K562 cells transfected with pTRE-ERG3 were subsequently used for SYBR Green RT–PCR analysis for determining fold changes in ERG and WNT11 mRNA levels in cells treated with (+) and without (−) dox over a 72-h period. Strong co-induction levels of ERG and WNT11 mRNA expression are displayed by black and gray bars, respectively. The induction levels were normalized to uninduced (without dox) cells. A representative SYBR Green RT–PCR assay of pTRE-ERG3 individual stable clones with strong ERG and WNT11 mRNA induction is shown. (b) K562 cells transfected with pTRE-ERG3 show elevated ERG protein levels upon dox induction. A representative western blot of using ERG antibody C20 in the tet-on ERG-inducible assay is shown. ERG protein levels are induced with the addition of dox (+) and uninduced without dox (−) in cells normally lacking ERG. (c) Long-term culture (8 days of dox stimulation) of ERG-induced cells resulted in an increase in WNT11, GATA-4 and TNFRSF25 mRNA expression in four individual clones. Relative mRNA expression levels were determined by duplicate SYBR Green assays in duplicate wells.

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To determine the effects of downstream targets with the ERG-inducible assay, we measured mRNA expression of all 17 putative ERG targets. WNT11 was the only co-induced gene with ERG mRNA after a 72-h induction. In ERG-induced cells, WNT11 was co-expressed in multiple individual clones in the 2–5-fold range (Figure 4a, gray bars). Furthermore, WNT11 mRNA expression was undetectable in untransfected K562 cells; thus, fold changes in WNT11 mRNA expression were entirely ERG-dependent.

A re-evaluation of 17 putative targets following prolonged growth (continuous dox stimulation for 8 days) of ERG-inducible cells was conducted. ERG mRNA expression doubled and WNT11 mRNA expression was upregulated by 15–19-fold (Figure 4c) when compared with 72h dox stimulation (Figure 4a). In addition, candidate target genes GATA-4 was upregulated 4–6-fold and TNFRSF25 was upregulated by twofold (Figure 4c). Thus, prolonged growth of ERG-induced cells not only enhanced upregulation of WNT11 but also influenced TNFRSF25 and GATA-4 transcripts. Other putative targets unaffected by these measures may require additional factors, or simply ERG alone cannot directly affect gene expression because of tight regulation or dysregulation, as is often the case in leukemias.

To determine if ERG bound the WNT11 promoter in vivo, 1.2kb of the WNT11 proximal promoter was cloned into a luciferase reporter vector, pGL3-basic. The 1.2-kb WNT11 promoter insert (denoted as pGL3–WNT11) was designed to include the oligonucleotide sequence spotted on the chip for ChIP-chip hybridization, the oligonucleotide sequence in the amplified product for quantitative PCR, and the significantly determined ETS-binding motifs. Transfection of pGL3–WNT11 into ERG-inducible cells reliably activated luciferase activity in response to ERG induction when compared with no dox conditions (Supplementary Figure 2b). We conclude that these results indeed confirm WNT11 is a direct target of ERG.

GSK-3β inhibition promotes growth advantage in ERG-induced cells

Owing to increased WNT11 transcripts prompted by ERG induction and promoter enrichment of several WNT genes, we hypothesized that ERG overexpression influenced the WNT pathway. Glycogen synthase kinase, GSK-3β, a multifaceted kinase central to WNT signaling, functions in a diverse manner through regulation of cell proliferation, differentiation and survival (Wu and Pan, 2009). GSK-3β inhibitors have become an attractive therapeutic option, as they has been shown to suppress tumorigenesis (Luo, 2009). A small-molecule GSK-3β inhibitor, BIO, can activate canonical WNT signaling (Soda et al., 2008) and suppress leukemia cell proliferation (Holmes et al., 2008). The ERG-inducible assay was utilized to study the proliferative growth effects in the presence and absence of GSK-3β inhibitor, BIO. Upon ERG induction, there were no proliferative advantages between the presence and absence of dox in ERG-inducible cells. The addition of BIO to ERG-induced cells, however, resulted in a proliferative growth advantage, whereas treatment of ERG-uninduced cells with BIO effectively suppressed proliferation (Figure 5a). The proliferative advantage was further evidenced by the greater number of viable cells in BIO-treated cells with ERG induction versus proliferative suppression of ERG-uninduced cells (Figure 5b). Thus, overexpression of ERG provokes a proliferative resistance to WNT agonist, BIO.

Figure 5.
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GSK-3β inhibitor, BIO, promotes a proliferative growth advantage in ERG-induced cells. (a) Proliferation was measured at 24, 48 and 72h by WST-1 assay. BIO (1.5μM)-treated K562 pTRE-ERG3 cells elicit a growth advantage in the ERG-induced (solid line, black circles) cells over the BIO-treated uninduced cells (solid line, white circles) at 72h. BIO untreated K562 pTRE-ERG3 cells with ERG induction (dashed line, black squares) and without ERG induction (dashed line, white squares) do not significantly differ in proliferation. The proliferative growth advantage elicited by the addition of BIO to ERG-induced cells compared with BIO-treated uninduced cells was observed in three independent stable clones over a 3-day period. The absorbance measurement values are averages (with standard error bars) of five wells per experimental treatment. Paired Wilcoxon's rank sum test was used to determine the statistical differences. (b) Determination of viable cell numbers by trypan blue exclusion with BIO treatment at 72h of K562 pTRE-ERG3 cells under dox-induced (black bars) and uninduced (gray bars) conditions. The viable cell numbers are displayed as averages with standard error bars. Student's paired t-test was used to determine the statistical difference. Asterisk (*) indicates statistically significant differences (P-value<0.05).

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ERG overexpression induces morphological transformation

WNT11 is well known for promoting morphological changes during early cell differentiation (Eisenberg et al., 1997) and morphological transformation of terminally differentiated cells (Uysal-onganer et al., 2010). This knowledge provided a focus point as to determine the functional consequences of ERG and WNT11 overexpression. ERG-inducible clones were cultured for a prolonged time in the presence and absence of dox stimulation. As early as 4 days of culture, a considerable fraction of ERG-induced cells underwent morphological shape changes from small round K562 cells to cells that were polarized and elongated with bidirectional protrusions (Figure 6a). These shape changes only occurred in the presence of dox, whereas in the absence of dox, the ERG-uninduced cells retained the native round and unpolarized morphology. The elongated cells with bidirectional protrusions and attached morphology were further verified by strong fluorescence of the ERG-induced cells (cloned into a tet-on red fluorescence tracking vector) versus absence of fluorescence of the ERG-uninduced cells. ERG-inducible cells were then seeded in equal cell numbers onto a six-well dish with grids for enumeration of cells polarized with protrusions. Cells polarized with protrusions peaked at 50–60 cells per field after 8 days of dox stimulation (Figure 6b). This observation was also evident in seven of the eight stably pTR-ERG3 transfected clones.

Figure 6.
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Co-expression of ERG and WNT11 induces morphological transformation. (a) Potent ERG-inducible clones were cultured with continuous dox stimulation for 8 days. Morphological shape changes from round unpolarized cells to polarized cells with protrusions began to appear as early as day 4 in the presence of dox (+dox), whereas in the absence of dox (−dox) cellular morphology remained unchanged. Visualization of the cellular shape changes at × 10 and × 20 magnification of both dox-treated (+dox) and dox-untreated (−dox) cells are shown. Due to the red fluorescence traceable tet-on system, ERG-induced shape changes could be visualized by strong fluorescence of the elongated cells as opposed to the absence of fluorescence of ERG-uninduced cells. (b) ERG-inducible cells with and without dox were seeded at an equal cell number on a 2-mm-grid six-well dish for enumeration of cells polarized with protrusions (at × 10 magnification). Prolonged growth of ERG-induced cells peaked at day 8 having 50–60 polarized cells with protrusions per field, whereas uninduced cells did not undergo shape changes. Bar graph is representative of experiments conducted with three K562 pTRE-ERG3 clones and depicts the density of polarized cells with protrusions in 10 fields on the x-axis, and the height depicts number of cells polarized with protrusions per field. (c) ERG-induced and -uninduced cells were treated with and without BIO (1.5μM) and analyzed in a similar manner as described above. BIO-treated cells strongly inhibited the elongation process of ERG-induced cells when compared with BIO-untreated cells. (d) To determine if cell morphological transformation was due to ERG induction of WNT11, WNT11 siRNA was utilized to knock down WNT11 transcript levels. Duplicate transfections of WNT11 and control siRNA were conducted with two individual K562 pTRE-ERG3 stable clones. Pre-stimulated cells (3 days dox stimulation) were transfected with WNT11 and control siRNA. Knockdown of WNT11 transcripts was reduced by 70%. Asterisk (*) indicates statistically significant differences (P-value<0.05). (e) Duplicate transfections of WNT11 and control siRNA were seeded equally by cell number onto a 2-mm-grid six-well dish. The number of polarized cells with protrusions was counted as described above. siRNA-mediated WNT11 knockdown significantly reduced the number of cells polarized with protrusions per field (mean of 4.5cells/field) when compared with control siRNA (mean of 6.3cells/field). Bar graph is representative of experiments conducted with two K562 pTRE-ERG3 clones and depicts the density of polarized cells with protrusions in 40 fields (80 fields in total were analyzed) on the x-axis, and the height depicts number of cells polarized with protrusions per field. Statistical significance was determined by the non-parametric Wilcoxon's rank sum test.

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We then tested the effect of BIO on morphological changes. The addition of BIO led to a strong inhibition of the elongation process in the ERG-induced cells. ERG-uninduced cells treated with BIO retained the round morphology. This result implied that the presence of BIO inhibited elongation (Figure 6c), while eliciting a growth advantage of ERG-induced cells, as shown in Figure 5.

Finally, to test whether WNT11 directed morphological transformation of ERG-induced cells, WNT11 siRNA was introduced into ERG-induced cells pre-stimulated for 72h. Overall, the transfection procedure reduced dramatically the number of polarized cells with protrusions compared with cells that did not undergo electroporation. Despite this effect, siRNA mediated knockdown of WNT11, mRNA knockdown of 70% (Figure 6d) reduced the number of cells polarized with protrusions per field when compared with the control siRNA at day 6 of dox stimulation (Figure 6e). The mean of cells polarized with protrusions per field was 4.5 and 6.3 cells per field for WNT11 and control siRNA, respectively (two-sided P-value=0.007) in a total of 80 fields counted. These results demonstrate that WNT11 is necessary to undergo elongation and attachment in the morphological transformation of ERG-induced cells.

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Discussion

Genetic aberrations of transcription factors required for normal hematopoiesis are frequently found in leukemia and contribute to transformation of hematopoietic progenitors through deregulated signaling (Look and O’Neil, 2007; Chiang et al., 2009). The ETS transcription factor ERG is overexpressed in subtypes of newly diagnosed T-ALL and AML patients. Overexpression of ERG mRNA expression was further correlated with poor prognosis; however, the downstream effects of deregulated mRNA expression are not yet understood (Baldus et al., 2007). Interestingly, patients with complex cytogenetics attribute aberrant ERG expression to recurring chromosomal abnormalities such as amplification at regions of the ERG loci (Baldus et al., 2004). Thus, ERG deregulated expression might have a pivotal role, as an upstream regulator, in acute leukemia. Herein, we explored the potential signaling pathways that ERG could regulate by ChIP-chip screening. This whole-genome screen identified WNT11, a non-canonical WNT gene, as a direct downstream target of ERG. Furthermore, enrichment of the WNT11 promoter was confirmed from ChIP of primary normal and leukemia bone marrow cells. Other putative targets that remain to be explored are TNFRSF25 and GATA-4. Upregulation of WNT11 is often coupled with the upregulation of GATA-4 in cardiomyocytes (Flaherty and Dawn, 2008) and the co-expression is mirrored in the ERG-induced cells; thus, further studies for validating this association are necessary.

In previous studies, avian embryonic stem cell differentiation into multiple blood cell phenotypes was shown to be dependent on WNT11 expression (Brandon et al., 2000). Thus, similar to ERG function, WNT11 has an early developmental role in normal hematopoiesis. Moreover, emerging evidence reveals that genes belonging to the WNT pathway, both canonical and non-canonical WNT genes, are aberrantly expressed in leukemia due to mutations (Reya and Clevers, 2005) or aberrant methylation (Gomez-Roman et al., 2007; Martin et al., 2009) that result in WNT cascade errors. Interestingly, synonymous to ERG, WNT11 mRNA expression and protein were elevated in high-grade prostate tumors (Zhu et al., 2004; Uysal-onganer et al., 2010) as well as implicated in breast cancer progression (Lin et al., 2007). A random sampling of a larger cohort of AML, T-ALL and normal bone marrow diagnostic material in this study demonstrated a nonlinear quantitative relationship between ERG mRNA expression and WNT11 mRNA expression, although co-expression of both genes was evident in AML and in normal samples. Moreover, the nonlinear correlation between ERG ChIP enrichment and WNT11 gene expression also appeared to follow different dynamics. As one study proposed, gene expression has a linear relationship with ChIP enrichment for actively transcribed genes, but the linear model does not fit a considerable fraction of non-actively transcribed genes. This may be a limitation in the ChIP method, or more likely due to the complex intermediate steps in accessing the chromosome (Guenther et al., 2007). Recent in vitro and in vivo ChIP-seq binding studies of ERG in prostate cells have suggested that chromatin accessibility is a major determining factor for promoter occupancy, although the correlation to gene expression was not addressed (Wei et al, 2010). Thus, we stipulate from these results that ERG overexpression, coupled with co-expression of WNT11, may potentially have a role in T-ALL and AML through deregulated non-canonical WNT signals.

Next, we modulated a central junction of the WNT pathway through GSK-3β inhibition. GSK-3β inhibition through BIO is of particular clinical interest, as the effects can preserve and support proliferation of normal hematopoietic stem cells through canonical WNT signaling (Sato et al., 2004). In contrast, GSK-3β inhibition by BIO suppressed proliferation of various leukemia cell lines, including K562 (Holmes et al., 2008). Interestingly, WNT11 has an opposing effect, for instance, in murine embryonic stem cells a repression of canonical WNT signals is reported with exogenous addition of WNT11 (Singla et al., 2006; Anton et al., 2007). In our results, the addition of BIO elicited a growth advantage in ERG-induced cells. This result may be due to ERG/WNT11 function overriding BIO effects (presumably canonical WNT signals) through stimulation of WNT non-canonical signals. Those signals, we propose, possibly occur through the Ca2+-dependent MAPK or PI3K-AKT pathways, as both pathways were shown to downregulate GSK-3β activity (Bikkavilli et al., 2008; Hu et al., 2009).

Finally, the potent morphological transformation of ERG-induced cells was unanticipated, yet plausible, according to a recent report describing significant association of ERG-positive tumors with WNT pathway signaling genes and epithelial-to-mesenchymal transition genes (Gupta et al., 2010). In our study, ERG-induced morphological shape changes also complement the initial global ChIP-chip analysis that determined cell adhesion as a significantly enriched biological theme (Table 2). Moreover, there is surmounting evidence of WNT11 as a morphogenic and cytoskeletal effector molecule (Zhou et al., 2007; Flaherty and Dawn, 2008; Lai et al., 2009; Uysal-onganer et al., 2010). Morphological transformation involving ERG gene activation of WNT11 may have a role in the bone marrow microenvironment. In the case of leukemia, high ERG-expressing cells producing morphogen WNT11 may create a niche in the bone marrow for resistant cells. Further investigations are warranted to determine the consequences of high ERG and WNT11 expression in the bone marrow microenvironment.

In conclusion, this genome-wide screen revealed WNT pathway as a major transcriptional network of ERG. Specifically, WNT11 emerged as the most prominent candidate of ERG. GSK-3β inhibition provoked a proliferative growth advantage in cells with ERG abundance and co-expression of WNT11, implying ERG's involvement in WNT signaling. This result suggests that for high-risk acute leukemia patients, characterized by high ERG mRNA expression, GSK-3β inhibitors as potential therapeutic options may be insufficient due to intrinsic ERG-induced resistance. Finally, morphogenic transformation of ERG-induced cells mediated by co-expression with WNT11 implicates a potential role for ERG- and WNT11-activating genetic programs that promote polarization and protrusions. This type of cellular response is a biological signal for cell migration and cell invasion by which these pathogenic mechanisms have already been proposed for ERG in prostate cancer (Klezovitch et al., 2008). Herein, these data collectively provide novel molecular components of ERG-directed cellular signals as a model for acute leukemia patients with high ERG expression and poor prognosis.

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Materials and methods

Cell culture and chemicals

Jurkat, KG1 and K562 cell lines were obtained from the German Resource Center for Biological Material, DSMZ (Braunschweig, Germany), and grown in RPMI media with 10% fetal bovine serum. All cell lines were cultured at 37°C in a 5% CO2 humidified chamber. BIO, a GSK-3β inhibitor, was purchased from Stem Cell Technologies (Vancouver, BC, Canada) and dissolved in DMSO.

Patient material

To conduct ChIP assays, we obtained untreated bone marrow aspirates from adult patients with newly diagnosed AML (n=5), T-ALL (n=1) and one healthy donor. Randomly selected diagnostic AML (n=30), T-ALL (n=30) and normal bone marrow (n=30) samples were obtained for the measurement of ERG and WNT11 mRNA expression. AML patients were treated at Charité, Campus Benjamin Franklin, Department of Hematology and Oncology, Berlin, Germany. T-ALL specimens were obtained from patients enrolled in the German Acute Lymphoblastic Leukemia Multicenter Study Group 07/03. Written informed consent was obtained from all patients and healthy volunteers, and the local ethics committee approved the study.

ChIP-chip

ChIP-chip was carried out as described in the NimbleGen Systems, Inc. (Madison, WI, USA) and Farnham (2009) methods. Briefly, Jurkat cells (1 × 109 cells) were crosslinked with formaldehyde. Following this, cells were suspended in lysis solution and DNA was fragmented to sizes ranging from 200 to 1000bp. The ERG-bound DNA was enriched with two ERG-specific antibodies targeting two different epitopes of the protein. The antibodies used for immunoprecipitation were rabbit Erg-1/2/3 C20 (epitope at C-terminus) and an equimolar mix of rabbit C20 and rabbit C17 (epitope corresponding to internal ERG protein) and non-specific IgG antibody as control (Santa Cruz Biotechnologies, Santa Cruz, CA, USA). Reverse crosslinking, DNA purification and ligation-mediated PCR (LM-PCR) amplification enabled to obtain the following experimental groups: enriched DNA by antibodies C20, C17/C20, IgG and total chromatin. Four promoter array hybridizations were conducted as follows: C20 enriched DNA (Cy5-label) was paired with total chromatin (Cy3-label) and referred to as ChIP-chip 1. The second hybridization (ChIP-chip 3) consisted of C20 enriched DNA (Cy5-label) and was paired with IgG-enriched DNA (Cy3-label). The second pair of hybridizations (ChIP-chips 2 and 4) include C17/C20 enriched DNA (Cy5-label) paired with total chromatin (Cy3-label) and C17/C20 enriched DNA (Cy5-label) paired with IgG-enriched DNA (Cy3-label). The hybridization was performed with a high-resolution promoter tiled array consisting of 50–75mer probes (770000) that represents 29000 annotated human transcripts (NimbleGen Systems, Inc.). Algorithms for Calculating Microarray Enrichment, ACME (Fred Hutchinson Cancer Research Center, Seattle, WA, USA), was used to determine potential genomic enriched regions, by sliding window analysis (using a window size of 2000bp) with Lowess normalized log2 (Cy5/Cy3) ratios. The calculated P-values were Benjamini–Hochberg corrected and a threshold was set at 90% (ACME bioinformatics software). For statistical analysis, significant gene annotations derived from hybridizations, ChIP-chips 1 and 3 were combined and are referred to as ChIP-chip I. Significant gene annotations from hybridizations and ChIP-chips 2 and 4 were combined and are referred to as ChIP-chip II. Only overlapping gene sets from ChIP-chips I and II were considered as candidate genes (Table 1). A detailed ChIP-chip description and complete NimbleGen promoter array data are available from the GEO database accession number GSE21495.

Databases

Databases used in our analyses were as follows: for gene promoter analyses, the Cold Spring Harbor Laboratory Transcriptional Regulatory Element Database, Swiss Institute of Bioinformatics Eukaryotic Promoter Database and Gene Regulation MATCH program were used to predict proximal promoter regions and predict conserved ETS-binding motif. DAVID Bioinformatics Resources 6.7 (Dennis et al., 2003; Huang et al., 2009) and Ingenuity Pathway Analysis programs were used to categorize biological pathways/themes of candidate gene lists.

ChIP of primary leukemia blasts

Primary bone marrow samples with sufficient material (cell numbers) to conduct ChIP assays were selected. Freshly prepared bone marrow aspirates from newly diagnosed AML patients, a T-ALL patient and a normal bone marrow donor were enriched for the mononuclear fraction by Ficoll-Hypaque density gradient (Amersham Pharmacia Biotech, Uppsala, Sweden). The following was performed for each donor: cell count was determined and cells were crosslinked with formaldehyde according to NimbleGen protocols. Immunoprecipitation was performed using 3μg of either C20 ERG antibody or control anti-IgG antibody to precipitate ERG-bound DNA complexes. A total of 2mg of protein per ChIP was used to perform two independent ChIPs for each antibody. Precipitated DNA templates were purified and amplified as described above.

Quantitative PCR of promoter regions

Quantitative PCR was performed to validate enrichment of candidate target promoter regions obtained from the ChIP-chip genome-wide screen. Proximal promoter primers within 2kb from the TSS were designed to include at least one of the estimated conserved ETS-binding motifs, 5-GGAA/T-3 and are described in Supplementary Table 1. SYBR Green (Invitrogen GmbH, Karlsruhe, Germany) based quantitative PCR was utilized to measure the relative abundance of enriched DNA to total chromatin. The cut-off for enriched target genes was set at twofold enrichment in both I and II ChIP groups versus controls IgG-enriched DNA and total chromatin.

Quantitative PCR of gene expression

Total RNA was purified from RNeasy plus (Qiagen GmbH, Hilden, Germany). First-strand complementary DNA (cDNA) synthesis was performed with AMV reverse transcriptase kit (Roche Diagnostics GmbH, Mannheim, Germany). Quantitative real-time–PCR (RT–PCR) was carried out in duplicate using SYBR Green master mix (Invitrogen GmbH). Amplifications were performed using the following conditions: 95°C, 10min; 40 cycles of 95°C, 30s; 55°C, 1min; 72°C, 30s. The comparative CT equation (2−ΔΔCT) was used to determine the relative expression levels of all genes used in this study under control and experimental conditions (primer pairs described in Supplementary Table 2). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was utilized as an internal control throughout this study. Multiplex RT–PCR to determine ERG mRNA expression was previously described (Bohne et al. 2009). WNT11 mRNA expression primer pairs and probe are described in Supplementary Information. Quantitative measurements of both genes in patient material were done using TaqMan Gene Expression Assays in duplicate wells (Applied Biosystems Inc., Foster City, CA, USA). WNT11 transcripts in patient material were considered ‘present’ with CT values less than or equal to27, were co-amplified with GUS as the house keeping gene and values shown are relative to WNT11 mRNA expression in KG1 cells.

siRNA-induced ERG knockdown

KG1 cells and K562 pTRE-ERG3-inducible clones were transfected according to AMAXA (Lonza Cologne AG, Cologne, Germany) as per the manufacturer's instructions, with ERG-specific siRNA (sih5ERG), or control siLuciferase (both siRNAs were kind gifts from Silence Therapeutics, Berlin-Buch, Germany), or On-Target plus siRNA WNT11 (Dharmacon, Denver, CO, USA). RNA knockdown levels were monitored by quantitative PCR. ERG protein levels were additionally monitored by standard western blotting techniques using antibody C20 for ERG detection.

Inducible expression constructs

ERG3 cDNA was synthesized by reverse transcription using Superscript III (Roche Diagnostics GmbH) with ERG isoform-specific primers as previously characterized (Bohne et al., 2009). A commercial ‘tet-on’ two-plasmid system (Clontech, Mountain View, CA, USA) was used to induce ERG expression in trans under the control of a tetracycline-inducible promoter. One vector, ptet-onAdvanced (pTet), expresses the transactivator protein only in the presence of an analog of tetracycline, doxycycline (referred to hereafter as dox, purchased from Clontech). An insert of ERG3 isoform was subcloned into a second vector, pTRE-Tight-BI-DsRed-Express (pTRE), which includes a RED fluorescent gene with a bidirectional promoter consisting of binding sites for transactivator protein. Vectors pTet (along with a linear puromycin marker) and pTRE-ERG3 were sequentially transfected into K562 cells using electroporation (Lonza Cologne AG, Cologne, Germany). K562 cells lack ERG mRNA expression and therefore ERG mRNA detection is entirely dependent on dox induction. Transfected K562 cells were maintained with puromycin at 1.25μg/ml (Merck, Nottingham, UK) and G418 at 800ng/ml (GIBCO, Invitrogen GmbH) to maintain double stable clones. Transfected bulk cells were then seeded to expand individual stable clones and examined for ERG induction by the addition of dox (1μg/ml). ERG induction was determined by red fluorescence detection and standard western blotting (using ERG-specific C20 antibody), and measurement of relative ERG mRNA expression by RT–PCR. For experimental controls, dox-untreated cells were cultured in parallel to dox-treated cells as uninduced status throughout each assay described in this study. Stable transfected clones displaying strong ERG mRNA expression upon dox induction were selected for further analyses. Stably transfected K562 cell derivatives in this study are referred to pTRE-ERG3. Stable clones are enumerated as CL1, CL2, CL3 and so on.

Luciferase reporter assays were conducted in the ERG-inducible expression assay. Essentially, WNT11 proximal promoter spanning +193 to -1042bp relative to TSS (~1.2kb) was cloned into pGL3-basic vector (Promega, Madisson, WI, USA). pRL-TK vector (kind gift from H Fechner lab, Department of Cardiology, Charité, Berlin, Germany) was used for the normalization of transfection efficiency and as control measurement of renilla luciferase activity. Vectors pGL3–WNT11 and pRL-TK were co-transfected into cells stimulated with and without dox. As control, pGL3-basic and pRL-TK were transfected individually in dox-stimulated cells. Firefly and renilla luciferase activities were measured with Dual-Luciferase Reporter Assay System (Promega).

Cell proliferation and enumeration

Cell proliferation was measured with WST-1 reagent according to the manufacturer's instructions (Roche Diagnostics GmbH). Briefly, cells for each time point were seeded in a 96-well plate with 3 × 104/well. K562 pTRE-ERG3 stable clones were cultured with and without dox for a 24-h period. Following this, pTRE-ERG3-transfected cells were treated with BIO for 3 days. Absorbance (450nm) of the reaction of WST-1 conversion to formazon dye was measured after 2h incubation with WST-1 at 24, 48 and 72h. The trypan blue dye exclusion test was used to determine the number of viable cells by cell counting with a hemocytometer.

Counting of polarized cells with protrusions involved seeding ERG-induced and uninduced cells at equal cell numbers onto a 2-mm-grid six-well dish (Nalge Nunc International, Rochester, NY, USA). Following dox stimulation up to 8 days, the suspended cell culture was gently removed for enumeration of cells polarized with protrusions at × 10 magnification. Experiments conducted for siRNA-mediated knockdown of WNT11 were counted in duplicate. To count cells in an even distribution of the culture dish surface area, culture dishes were divided into 3–4 sections, and 10 fields per section were counted by two persons. In addition, a blinded system was used for cell enumeration to ensure reproducibility.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

This study was supported by a grant from the Deutsche Krebshilfe (Max Eder Nachwuchsförderung) to CDB. We thank Dr Martin Neumann (Department of Hematology and Oncology, Charité, Berlin, Germany) for his critical reading of the manuscript. We also thank Dr Anja Kühl (Research Center ImmunoSciences, Charité, Berlin, Germany) for helpful advice and fluorescence microscopy assistance.

Supplementary Information accompanies the paper on the Oncogene website