Abstract
Background/aim: Ewing's sarcoma is an aggressive pediatric malignancy driven by specific chromosomal translocations, predominantly leading to the oncogenic fusion protein EWS-FLI1. Runt-related transcription factors 1 and 2 (RUNX1 and RUNX2), which are key transcription factors involved in osteoblastic differentiation and skeletal morphogenesis, are not expressed in Ewing's sarcoma. Our study aimed to elucidate the relationship between the RUNX genes and EWS-FLI1. Materials and methods: We used Ewing's sarcoma cell lines (A673 and NCR-EW2). EWS-FLI1 knockdown was achieved using shRNA, while inducible expression of RUNX1, RUNX2, EWS-FLI1, or EWS-ERG was performed using a Tet-on system. Gene and protein expression levels were quantitatively assessed by qPCR and Western blot analysis, respectively. Cell growth was evaluated using spheroid formation and cell viability assays. Truncated RUNX1 or RUNX2 constructs were employed to identify functional domains, with their intracellular localization confirmed by immunostaining. Results: We observed low expression of RUNX1 and RUNX2 in Ewing's sarcoma cells. Notably, EWS-FLI1 knockdown increased RUNX2 gene expression. Conversely, inducible expressions of either RUNX1 or RUNX2 suppressed the expression of EWS-FLI1 target genes and inhibited cell proliferation and spheroid formation. Additionally, the RUNT domain of RUNX1 and RUNX2 was not essential for their inhibitory effect on EWS-FLI1 target gene expression. Importantly, both EWS-FLI1 and EWS-ERG down-regulated RUNX2 gene expression in hTert-RPE1 cells. Conclusion: EWS-FLI1 suppresses RUNX2 gene expression in Ewing's sarcoma cells, and re-expressing RUNX2 inhibits cell growth. These findings suggest that RUNX2 has a negative impact on the oncogenic process in Ewing's sarcoma.