β-Catenin in desmoid-type fibromatosis: deep insights into the role of T41A and S45F mutations on protein structure and gene expression

Abstract

Desmoid-type fibromatosis (DF) is a rare mesenchymal lesion with high risk of local recurrence. Specific β-catenin mutations (S45F) appeared to be related to this higher risk compared to T41A-mutated or wild-type (WT). We explored the influence of both mutations and WT on structure stability and affinity of β-catenin for α-catenin and the pattern of gene expression that may influence DF behavior. Using 33 surgically resected primary DFs harboring T41A (n = 14), S45F (n = 10), or WT (n = 9), we performed a comparative molecular analysis by protein/protein interaction modeling, gene expression by DASL microarrays, human inflammation gene panel, and assessment of immune system-based biomarkers by immunohistochemistry. Mutated proteins were more stable than WT and formed a weaker complex with α-catenin. Consensus unsupervised gene clustering revealed the presence of two DF group-mutated (T41A + S45F) and WT (P = 0.0047). The gene sets 'Inflammatory-Defense-Humoral Immune Response' and 'Antigen Binding' were significantly enriched in T41A. The deregulation of 16 inflammation-related genes was confirmed. Low numbers of T cells and tumor-associated macrophages (TAM) infiltrating the tumors and low/absent PD-1/PD-L1 expression were also identified. We demonstrated that mutated DFs (T41A or S45F) and WT are two distinct molecular subgroups with regard to β-catenin stability, α-catenin affinity, and gene expression profiling. A different inflammation signature characterized the two mutated groups, suggesting mediation either by T41A or by S45F. Finally, all mutated cases showed a low number of TIL and TAM cells and a low or absent expression of PD-1 and PD-L1 consistent with β-catenin activation insensitive to checkpoint blockade.

Keywords: desmoid-type fibromatosis; gene expression; modeling; β-catenin mutation.

Figure 1

Wild‐type (WT) and mutated β‐catenins have different thermodynamic stability and affinity for α‐catenin. (A) Three‐dimensional structure of the β‐catenin WT protein. The different domains of β‐catenin are shown in different colors (same for 3D model and diagram): N‐terminal domain (NTD), blue; Armadillo repeated units, orange; C‐terminal domain (CTD), purple. (B) Overall and (C) zoomed view of an equilibrated MD snapshot of the WT β‐catenin/α‐catenin complex. The proteins are visualized in ribbon style and colored as follows: α‐catenin, cornflower blue; β‐catenin, orange. Oxygen water atoms are represented as red spheres, while chlorine and sodium are depicted as green and purple spheres, respectively. (D) Superposition of MD‐equilibrated snapshots of WT (cornflower blue) and the T41A (orange)‐mutant β‐catenin in complex with α‐catenin. (E) Superposition of MD‐equilibrated snapshots of WT (cornflower blue) and the S45F (purple)‐mutant β‐catenin in complex with α‐catenin.

Figure 2

Mutated and wild‐type (WT) β‐catenin desmoid‐type fibromatosis (DFs) have different gene expression profiles. (A) Upper panel: consensus unsupervised clustering analysis of the gene expression of 33 DF samples (nine WT and 24 mutated); the heatmap depicts the consensus matrix imposing the presence of two clusters on the dataset. The values range from 0 (white, samples do not cluster together) to 1 (blue, samples showing high clustering affinity); lower panel: distribution of WT and mutated DF samples in the two clusters. (B) Volcano plot of log₂ (fold changes) versus −log₁₀ P‐value. The volcano plot shows transcriptional differences between the two groups of samples identified by consensus unsupervised clustering analysis. The dashed lines denote the FDR < 0.01 cutoff. (C, D) The list of genes, down (119) and up (636), in sporadic DFs (n = 14) as compared to normal samples/solitary fibrous tumors (n = 11) (31), when used as custom gene set in GSEA, identified in WT CTNNB1 cases (panel C) a significant enrichment of genes present in normal/solitary fibrous tissues, and in mutated CTNNB1 cases (panel D) a significant enrichment of genes present in sporadic DF.

Figure 3

Biological pathways modulated by CTNNB1 mutational status. Bubble plot of gene sets significantly enriched in: (panel A) CTNNB1‐mutated DF as compared to WT DF; (panel B) CTNNB1 T41A DFs as compared to S45F. An overview of GSEA‐enriched networks is depicted. The x‐axis indicates the z‐score for each term, while the y‐axis is the negative logarithm of the adjusted P‐value. The area of the displayed circles is proportional to the number of genes assigned to each term. DF, desmoid‐type fibromatosis; WT, wild‐type.

Figure 4

T cells and tumor‐associated macrophages (TAM) are rare in T41A‐ or S45F‐mutated desmoid‐type fibromatosis. In the tumor proliferation (A), a low number of CD3⁺ T cells (B) and CD163⁺ tumor‐associated macrophages (C) were distributed around the vessels. In occasional small lymphoid aggregates (D), CD20⁺ B cells (E), CD3⁺ T cells (F), and EZH2⁺ cells (G) were present. Few PD‐1‐decorated lymphocytes were restricted to lymphoid aggregates (H), and no immunolabeled PD‐L1 tumor‐associated inflammatory cell (I) was present.