Deciphering the ways in which somatic mutations and germline susceptibility variants cooperate to promote cancer is challenging. Ewing sarcoma is characterized by fusions between EWSR1 and members of the ETS gene family, usually EWSR1-FLI1, leading to the generation of oncogenic transcription factors that bind DNA at GGAA motifs1, 2, 3. A recent genome-wide association study4 identified susceptibility variants near EGR2. Here we found that EGR2 knockdown inhibited proliferation, clonogenicity and spheroidal growth in vitro and induced regression of Ewing sarcoma xenografts. Targeted germline deep sequencing of the EGR2 locus in affected subjects and controls identified 291 Ewing-associated SNPs. At rs79965208, the A risk allele connected adjacent GGAA repeats by converting an interspaced GGAT motif into a GGAA motif, thereby increasing the number of consecutive GGAA motifs and thus the EWSR1-FLI1–dependent enhancer activity of this sequence, with epigenetic characteristics of an active regulatory element. EWSR1-FLI1 preferentially bound to the A risk allele, which increased global and allele-specific EGR2 expression. Collectively, our findings establish cooperation between a dominant oncogene and a susceptibility variant that regulates a major driver of Ewing sarcomagenesis.
At a glance
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- Supplementary Figure 1: Expression pattern of EGR2 and ADO in normal tissues relative to Ewing sarcoma. (197 KB)
The normal-body atlas consisted of 353 microarrays representing 63 individual tissue types (GSE3526). Gene expression levels are shown as the mean and s.e.m. of n ≥ 3 samples per tissue type. The normal-body atlas and the Ewing sarcoma (EwS) data (GSE34620) were normalized simultaneously by RMA using custom brainarray CDF (v18, ENTREZG). All microarray data were generated on Affymetrix HG-U133Plus2.0 arrays.
- Supplementary Figure 2: eQTL analyses across tissue types identify Ewing sarcoma-specific correlations of EGR2 and ADO expression with the risk-allele (G) at rs1848797. (444 KB)
Data are shown as medians (horizontal bars) with ranges for the 25th−75th percentile (box) and 10th−90th percentile (whiskers). Outliers are depicted as dots. P values determined via linear regressions. EwS, Ewing sarcoma; AML, acute myeloid leukemia; LCL, lymphoblastoid cell lines.
- Supplementary Figure 3: Analysis of EGR2 and ADO expression in Ewing sarcoma cell lines after knockdown of EWSR1-FLI1. (84 KB)
A673, SK-N-MC, EW7 and POE cells were transfected either with non-targeting control siRNA or siRNA targeting EWSR1-FLI1. Gene expression was assessed 48 h thereafter by qRT-PCR. FC, fold change. Data are shown as the mean and s.e.m.; n ≥ 5 independent experiments. Two-tailed unpaired Student’s t-test; ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001.
- Supplementary Figure 4: Knockdown of EGR2 reduces clonogenic growth, cell cycle progression, and viability of Ewing sarcoma cells. (290 KB)
(a) Analysis of EGR2 protein expression by immunoblot in Ewing sarcoma cell lines (loading control: β-actin). The neuroblastoma cell line SK-N-SH served as negative control. (b) Analysis of EGR2 protein expression by immunohistochemistry in tumors of xenografted Ewing sarcoma cell lines (A673, TC-71 and SK-ES1), the alveolar rhabdomyosarcoma cell line SJ-RH30, and the neuroblastoma cell line IMR-32, and comparison with EGR2 mRNA expression levels as determined by Affymetrix HG-U133Plus2.0 arrays (GSE36133). Scale bars = 200 µm. (c) qRT-PCR analysis of knockdown efficacy of siRNAs used to silence EGR2 or ADO (48 h after transfection). Data are shown as the mean and s.e.m.; n ≥ 4 independent experiments. (d) Analysis of clonogenic growth after seeding at low-density and serial re-transfection with siRNA every four days. Cells were fixed 9–14 days after seeding and individual colonies were colorized with crystal violet. (e) Analysis of cell cycle phases by PI staining 96 h after transfection with siRNA. EGR2 knockdown reduces the percentage of cells in S phase (P < 0.01 for A673, SK-N-MC, and POE; P = 0.12 for EW7), while increasing the percentage in sub G1 phase (all P < 0.0001). (f) Analysis of apoptosis by Annexin-V-staining 96 h after transfection with siRNA. Data in d-f are shown as the mean and s.e.m. of results obtained with two different siRNAs for EGR2 and three different siRNAs for ADO as displayed in c; n ≥ 3 independent experiments. Two-tailed unpaired Student’s t-test; ns, not significant; *** P < 0.001.
- Supplementary Figure 5: EGR2 is a downstream component of the FGF pathway in Ewing sarcoma. (121 KB)
(a) Scatter-dot plot and medians (horizontal bars) of EGFRs (gray) and FGFRs (red) expression levels in n = 117 primary Ewing sarcoma tumors (GSE34620). (b) Analysis of proliferation of Ewing sarcoma cell lines with a Resazurin assay. Cells were seeded in RPMI 1640 medium with 0.5–1% FCS and stimulated with the indicated growth factor (GF) concentrations for 72 h. Data are shown as the mean and s.e.m.; n ≥ 6 experiments. (c) Analysis of EGR2 induction by qRT-PCR in Ewing sarcoma cell lines after incubation with either EGF or bFGF (both 10 ng/ml). The EGFR expressing MDA-MB-231 breast cancer cell line served as a positive control for EGF action. Data are shown as the mean and s.e.m.; n ≥ 3 independent experiments. Two-tailed unpaired Student’s t-test, ** P < 0.01, *** P < 0.001.
- Supplementary Figure 6: Targeted germline deep sequencing of principal-component analysis (PCA)-matched Ewing sarcoma cases and controls. (177 KB)
(a) PCA-clustering of the selected core population (dashed box) for sequencing. (b) Analysis of target-region coverage after mapping and base quality filtering of reads (MQ20 and BQ20). (c) Analysis of average nucleotide coverage (high-quality reads) of the chr10 target-region across all samples. The median nucleotide coverage is reported (217X).
- Supplementary Figure 7: Schematic illustration of the workflow for next-generation sequencing and variant analysis. (299 KB)
AAR, alternative allele ratio.
- Supplementary Figure 8: Genomic coordinates, evolutionary sequence conservation, aligned DNA sequences and homology of EGR2 enhancer elements. (456 KB)
Mammal Cons, 46 vertebrates basewise conservation from the UCSC genome browser60; GERP, Genomic Evolutionary Rate Profiling of 30 vertebrate species from the UCSC genome browser; MultiZ Align, multiple sequence alignment from the UCSC Genome browser; MSE, Myelinating Schwann cell Enhancer; BoneE, Bone Enhancer. Sequence alignment of murine and human DNA sequences was carried out using Clustal Ω (v1.2.0)45. Asterisks indicate homologous nucleotides.
- Supplementary Figure 9: Genomic coordinates, epigenetic profile and reference sequence of the mSat1 locus. (103 KB)
GGAA repeats are underlined by arrows. The reported numbers of GGAA motifs correspond to the reference sequence (hg19).
- Supplementary Figure 10: Validation of ChIP efficiency by qRT-PCR in the Ewing sarcoma cell line MHH-ES1 (A/T at rs79965208). (67 KB)
A described CCND1 EWSR1-FLI1 binding site was used as positive control47, and an intronic CCND1 locus (intron 2) served as negative control. Data are shown as the mean and s.d.
- Supplementary Figures (2,386 KB)
Supplementary Figures 1–11