Clinical and biochemical function of polymorphic NR0B1 GGAA-microsatellites in Ewing sarcoma: a report from the Children's Oncology Group

Abstract

Background: The genetics involved in Ewing sarcoma susceptibility and prognosis are poorly understood. EWS/FLI and related EWS/ETS chimeras upregulate numerous gene targets via promoter-based GGAA-microsatellite response elements. These microsatellites are highly polymorphic in humans, and preliminary evidence suggests EWS/FLI-mediated gene expression is highly dependent on the number of GGAA motifs within the microsatellite.

Objectives: Here we sought to examine the polymorphic spectrum of a GGAA-microsatellite within the NR0B1 promoter (a critical EWS/FLI target) in primary Ewing sarcoma tumors, and characterize how this polymorphism influences gene expression and clinical outcomes.

Results: A complex, bimodal pattern of EWS/FLI-mediated gene expression was observed across a wide range of GGAA motifs, with maximal expression observed in constructs containing 20-26 GGAA motifs. Relative to white European and African controls, the NR0B1 GGAA-microsatellite in tumor cells demonstrated a strong bias for haplotypes containing 21-25 GGAA motifs suggesting a relationship between microsatellite function and disease susceptibility. This selection bias was not a product of microsatellite instability in tumor samples, nor was there a correlation between NR0B1 GGAA-microsatellite polymorphisms and survival outcomes.

Conclusions: These data suggest that GGAA-microsatellite polymorphisms observed in human populations modulate EWS/FLI-mediated gene expression and may influence disease susceptibility in Ewing sarcoma.

Figure 1. Flow diagram of COG study AEWS0031 patient samples included for GGAA-microsatellite sequencing and clinical analysis.

Figure 2. GGAA-microsatellite organization at the NR0B1 locus.

(A) Using available single nucleotide polymorphism (SNP) data from the CEU reference population (northern and western European decent) of the International HapMap Project , the NR0B1 GGAA-microsatellite is identified within a defined haplotype block. (B) For the NR0B1 locus, the GGAA-microsatellite is located approximately 1.5 kb upstream of the transcriptional start site (TSS) and is characterized a variable number of contiguous GGAA motifs, partitioned by single adenosine base substitutions. Sequence characteristics of interest include the total number of GGAA motifs, the total number of contiguous segments and longest consecutive GGAA segment. Figure panel adapted from .

Figure 3. NR0B1 GGAA-microsatellites are polymorphic in Ewing sarcoma tumors with an allelic distribution different than that of white European and African controls.

(A) Principal components analysis comparing unique microsatellite haplotypes in tumor samples and white European controls demonstrate three principal sequence clusters, with a high-degree of overlap between the two populations. (B) Histogram plots comparing the distribution frequency of NR0B1 GGAA-microsatellite haplotypes in tumors and white European and African controls. Despite the overlapping PCA analysis, an enrichment of haplotypes containing 21–25 and 56–60 GGAA motifs was observed in tumor samples. Relative to white Europeans, a depletion of haplotypes containing 16–20 GGAA motifs was also noted in tumors. (C) Cumulative density plots for each study population similarly demonstrate the enrichment of haplotypes containing 21–25 and 56–60 GGAA motifs in tumors. The distribution of these haplotypes in tumors is significantly different from both white Caucasian and African populations (KS test, p<0.001). (D) Stratifying haplotypes according to the major sequence types identified in the PCA demonstrates that intermediate (3 segment) GGAA-microsatellites are more enriched in tumors and larger multisegment haplotypes (>3 segments) were also more enriched compared to white Europeans, although markedly less than Africans. Control white European and African population data from .

Figure 4. GGAA-microsatellites sequence characteristics after whole genome amplification (WGA).

Microsatellites were sequences after WGA and compared to unamplified DNA.

Figure 5. EWS/FLI-mediated gene expression is highly variable across various GGAA-microsatellite length polymorphisms.

(A) Polymorphic NR0B1 GGAA-microsatellites from white European and African subjects were cloned into luciferase reporter vectors and co-transfected with EWS/FLI into 293 EBNA cells. A bimodal pattern of gene expression was observed, with greatest expression in constructs with 24 GGAA motifs and a lesser peak in constructs with 58 GGAA motifs. (B) A similar bimodal trend was observed using synthetic GGAA constructs identically cloned into the same luciferase reporter construct. (C) In patient derived Ewing sarcoma cell lines, RT-PCR quantified NR0B1 mRNA expression was also maximal in cell lines containing an NR0B1 microsatellite containing 24–26 GGAA motifs. (D) In primary Ewing sarcoma tumors, normalized NR0B1 transcript levels were lowest in tumors with NR0B1 GGAA-microsatellites containing 17–18 GGAA motifs, which was significant less than tumors with microsatellites containing 23–26 GGAA motifs (p = 0.04).

Figure 6. NR0B1 GGAA-microsatellite polymorphisms do not influence event free survival (EFS) in Ewing sarcoma patients.

(A) EFS was compared in 112 patients from AEWS0031 based on the presence of absence of at least one NR0B1 GGAA-microsatellite allele containing 22–27 GGAA motifs. This allele type was chosen based on the pattern of alleles present in tumor samples and the maximal EWS/FLI-mediated gene expression supported by alleles of this length category. (B) EFS was similarly assessed based on the presence of one or both alleles containing 22–27 GGAA motifs. Additional subgroup analyses were also performed in males (C) and females (D) and in patients receiving standard (E) or compressed (F) therapy.

Figure 7. Model of GGAA-microsatellite polymorphism contributions to Ewing sarcoma susceptibility in African and white European populations.