Differentially Methylated Regions in Desmoid-Type Fibromatosis: A Comparison Between CTNNB1 S45F and T41A Tumors

Abstract

Introduction: The majority of desmoid-type fibromatosis (DTF) tumors harbor a β-catenin mutation, affecting specific codons in CTNNB1 exon 3. S45F tumors are reported to have a higher chance of recurrence after surgery and more resistance to systemic treatments compared to wild-type (WT) and T41A tumors. The aim of this pilot study was to examine the genome-wide DNA methylation profiles of S45F and T41A mutated DTF, to explain the observed differences in clinical behavior between these DTF subtypes.

Material and methods: Genome-wide analysis of DNA methylation was performed using MeD-seq on formalin-fixed, paraffin-embedded primary DTF samples harboring a S45F (n = 14) or a T41A (n = 15) mutation. Differentially methylated regions (DMRs) between S45F and T41A DTF were identified and used for a supervised hierarchical cluster analysis. DMRs with a fold-change ≥1.5 were considered to be differentially methylated and differences between S45F and T41A tumors were quantitatively assessed. The effect of DMRs on the expression of associated genes was assessed using an independent mRNA expression dataset. Protein-protein interactions between WT β-catenin and mutant variants and DNA methyltransferase 1 (DNMT1) were examined by immunoprecipitation experiments.

Results: MeD-seq analyses indicated 354 regions that displayed differential methylation. Cluster analysis yielded no distinct clusters based on mutation, sex, tumor site or tumor size. A supervised clustering based on DMRs between small (≤34 mm) and large (>87 mm) DTF distinguished the two groups. Only ten DMRs displayed a fold change of ≥1.5 and six of them were found associated with the following genes: NLRP4, FOXK2, PERM1, CCDC6, NOC4L, and DUX4L6. The effects of DMRs on gene expression yielded a significant difference (p < 0.05) in the expression between S45F and T41A for CCDC6 and FOXK2 but not for all Affymetrix probe-sets used to detect these genes. Immunoprecipitations did not reveal an association of WT β-catenin or mutant variants with DNMT1.

Conclusion: This study demonstrated that S45F and T41A DTF tumors did not exhibit gross differences in DNA methylation patterns. This implies that distinct DNA methylation profiles are not the sole determinant for the divergent clinical behavior of these different DTF mutant subtypes.

Keywords: DNA methylation; aggressive fibromatosis; epigenomics; soft tissue sarcomas; β-catenin.

Figure 1

Supervised hierarchical clustering based on differentially methylated regions (DMRs) between S45F and T41A mutated DTF tumors together with clinicopathological features: sex, tumor site, age, and tumor size. The heat map depicts the methylation in 354 DMRs, including all fold-changes and excluding DMRs present on sex chromosomal regions, in S45F and T41A DTF samples. The cluster tree on top indicates distinct subgroups of DTF samples. Grouping, however, is not based on CTNNB1 mutation type (T41A or S45F) nor on clinicopathological parameters such as sex, tumor site, age, or tumor size.

Figure 2

Read counts for DMRs in individual S45F and T41A DTF. Plots showing the normalized read counts for selected DMRs (fold-change ≥1.5) associated with NLRP4, FOXK2, PERM1, CCDC6, NOC4L and DUX4L6. The whiskers represent the minimum and maximum read counts. Dots (S45F) or squares (T41A) indicate individual data points, the horizontal line designates the median level. A Mann Whitney U test was used to assess statistical significance.

Figure 3

DNMT1 is not co-precipitated with wild-type or mutant CTNNB1 (β-catenin). HEK293T cells were transfected with plasmids driving the expression of FLAG-tagged wild-type β-catenin (WT) or FLAG-tagged mutant versions of β-catenin (T41A; S45P; Exon 3 deletion mutant; K335I). As a control cells were transfected with the empty vector. At 48-h post-transfection, cell lysates were prepared from which the FLAG-tagged β-catenin variants were immunoprecipitated. Western Blot analysis was used to examine DNMT1, β-catenin and β-actin protein expression in the total lysates and immunoprecipitates.