Transcriptional targets and protein interactions of NKX2-2 in Ewing sarcoma

Ewing sarcoma is a bone-associated malignancy of children and adolescents caused by EWS/FLI, an oncogenic transcription factor encoded by a chromosomal translocation. EWS/FLI causes massive transcriptional dysregulation. A critical upregulated target, NKX2-2, is a homeodomain transcription factor required for Ewing sarcomagenesis; however, its specific role in this disease is unknown. We addressed this question using a twofold approach. First, using RNA sequencing, we found that NKX2-2 represses genes important for cell adhesion and ECM organization. We also show that it inhibits mesenchymal features of Ewing sarcoma cells: actin stress fiber organization, focal adhesion assembly, cell spreadingâ€"phenocopying EWS/FLI knockdown. NKX2-2-depleted cells also display increased cell adhesion and migration. Finally, we show that NKX2-2 and ZEB2 mediate anti-mesenchymalization and anti-epithelialization programs, respectively, in Ewing sarcoma to keep cells in a partially undifferentiated state. Second, we show that NKX2.2 binds MTG16 in an interaction that is disrupted by Notch. Presumably, these proteins comprise a repressor complex given the proper cellular context. Furthermore, NKX2.2 is capable of oligomerization; interestingly, both heterotypic and homotypic interactions are mediated by the transcriptional activation domain of NKX2.2. While the biological significance of the NKX2.2-MTG16 interaction is currently unknown, we propose that investigating this complex may be tractable in pancreatic β-islet cells. Importantly, defining the transcriptional targets and elucidating the molecular interactions of critical transcription factors in Ewing sarcoma, as well as in other cancers, may more fully define their function.