NR4A3 fusion proteins trigger an axon guidance switch that marks the difference between EWSR1 and TAF15 translocated extraskeletal myxoid chondrosarcomas

Abstract

Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma histotype with uncertain differentiation. EMC is hallmarked by the rearrangement of the NR4A3 gene, which in most cases fuses with EWSR1 or TAF15. TAF15-translocated EMC seem to feature a more aggressive course compared to EWSR1-positive EMCs, but whether the type of NR4A3 chimera impinges upon EMC biology is still largely undefined. To gain insights on this issue, a series of EMC samples (7 EWSR1-NR4A3 and 5 TAF15-NR4A3) were transcriptionally profiled. Our study unveiled that the two EMC variants display a distinct transcriptional profile and that the axon guidance pathway is a major discriminant. In particular, class 4-6 semaphorins and axonal guidance cues endowed with pro-tumorigenic activity were more expressed in TAF15-NR4A3 tumors; vice versa, class 3 semaphorins, considered to convey growth inhibitory signals, were more abundant in EWSR1-NR4A3 EMC. Intriguingly, the dichotomy in axon guidance signaling observed in the two tumor variants was recapitulated in in vitro cell models engineered to ectopically express EWSR1-NR4A3 or TAF15-NR4A3. Moreover, TAF15-NR4A3 cells displayed a more pronounced tumorigenic potential, as assessed by anchorage-independent growth. Overall, our results indicate that the type of NR4A3 chimera dictates an axon guidance switch and impacts on tumor cell biology. These findings may provide a framework for interpretation of the different clinical-pathological features of the two EMC variants and lay down the bases for the development of novel patient stratification criteria and therapeutic approaches. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Keywords: EWSR1; NR4A3; TAF15; axon guidance; extraskeletal myxoid chondrosarcomas; sarcoma; transcriptional profile.

Figure 1

Transcriptome profiling of EMC. (A) Principal component analysis of the transcriptome of EWSR1‐NR4A3 (red) and TAF15‐NR4A3 (blue) rearranged EMC. (B) Z‐Score normalized heat map of the expression values (log2 transformed) of the top 500 differentially expressed genes in TAF15‐NR4A3 and EWSR1‐NR4A3 EMC. Sample (top) and gene (left) dendrograms are shown. Sample ID number and NR4A3 fusion partner are indicated. The color bar indicates the Z‐score and reflects the relative gene expression level, from blue (low), white (medium) to red (high). Gene cluster 1 consists of the genes overexpressed in EWSR1‐NR4A3 EMC; gene cluster 2, the genes overexpressed in TAF15‐NR4A3 EMC. (C) Log2 fold‐change of the axon guidance molecules differentially expressed in the two EMC variants. Black and grey bars indicate genes underexpressed and overexpressed, respectively, in TAF15‐NR4A3 versus EWSR1‐NR4A3 tumors.

Figure 2

Representative EMC immunostainings representative immunostainings for Nestin, Semaphorin 4D, Synaptophysin and Reelin in a set of EWSR1‐NR4A3 (top) and TAF15‐NR4A3 (bottom) EMC. Case ID number is indicated. Magnification: ×100.

Figure 3

Transcriptome profiling of E‐N, T‐N and NR4A3 cell models. (A) Top: Representative phase‐contrast images showing colony formation in soft agar of tBJ/ER cells engineered to express control empty vector (Ctrl), NR4A3, E‐N or T‐N. Magnification: ×100; scale bar = 200 μm. The plot (bottom) shows mean number of colonies >30 μm ± SE (black bars) and mean colony size ± SE (gray bars) per ×100 magnification field at day 8 post‐plating. (B) PCA of the transcriptome of NR4A3 (black), E‐N (red) and T‐N (blue) cell models. (C) Z‐score normalized heat map of the top 500 differentially expressed genes in T‐N versus E‐N cells. Sample (top) and gene (left) dendrograms are shown. Color‐coding is as in Figure 1. Gene cluster 1 consists of the genes overexpressed in E‐N; gene cluster 2, genes overexpressed in T‐N. (D) Network integration analysis of genes differentially expressed in both EMC (TAF15‐NR4A3 versus EWSR1‐NR4A3) and in cell models (T‐N versus E‐N). Axon guidance‐associated GO biological processes are highlighted in blue. (E) Heat map and hierarchical clustering for the axon guidance molecules that are differentially expressed in T‐N versus E‐N. NR4A3 co‐clusters with E‐N. (F) Plot showing the modulation in tBJ/ER T‐N versus E‐N cells (black bars) of the axon guidance cues that were detected as statistically differentially expressed in TAF15‐NR4A3 versus EWSR1‐NR4A3 EMC (grey bars). *Genes whose modulation (Log2 fold‐change) is statistically significant also in cell models (p < 0.05).

Figure 4

NR4A3 chimeras differentially bind the SEMA3C promoter and display diverse protein expression levels. (A) Schematic representation of the putative NR4A3 binding site identified by MatInspector on the human SEMA3C regulatory region. The NBRE‐like sequence and genomic coordinates are shown (human genome assembly GRCh38/hg38; chromosome 7). Arrows indicate the primers used in ChAP‐qPCR assays. (B) ChAP‐qPCR results of the binding of NR4A3 and NR4A3 chimeras to the indicated SEMA3C regulatory region. Relative enrichment for the SEMA3C NBRE target region in tBJ/ER cells expressing Strep‐tagged NR4A3, E‐N or T‐N versus negative control (empty vector, Ctrl) is shown. Relative enrichment indicates the amount of SEMA3C‐specific precipitated DNA normalized to the total input chromatin, with Ctrl set to 1. (C) Representative immunoblot for NR4A3 in tBJ/ER cells engineered to express EWSR1‐NR4A3, TAF15‐NR4A3, NR4A3, or control empty vector (Ctrl). The blot was hybridized with an anti‐N‐terminus MoAb (clone H7833). *Nonspecific band. (D) Relative expression levels of NR4A3‐related mRNAs (±SE) assessed by RT‐qPCR of the cells shown in (C).