Bledsoe, Krista L.

Abstract Ewing sarcomas are aggressive, poorly differentiated pediatric tumors of bone and soft tissue. Ewing sarcoma affects one to three people per million every year and is the second most common pediatric bone malignancy after osteosarcoma. Approximately 85% of Ewing sarcomas harbor the EWS-FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS-FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS-FLI binds the osteogenic transcription factor RUNX2, and prevents osteoblast differentiation. RUNX3 is highly homologous to RUNX2 and participates in the mesenchymal-derived bone formation. RUNX3 has been described as both a tumor suppressor and an oncogene in different tumor types. In this study, we investigated the role of RUNX3 in Ewing Sarcoma. RUNX3 was detected in all Ewing sarcoma cells examined (7/7 cell lines, and 4/4 primary tumors), whereas RUNX2 was detected in only 73% of these specimens. Immunoprecipitation experiments revealed that RUNX3 binds to EWS-FLI via its Runt domain, which is >91% identical to the Runt domain in RUNX2. The interaction between RUNX3 and EWS-FLI in cell nuclei was confirmed by immunofluorescence. Moreover, EWS-FLI interactions prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. To determine the role of RUNX3 in Ewing sarcoma cell growth, we stably suppressed RUNX3 expression in the Ewing sarcoma cell line A673 with shRNAs. In anchorage independent growth assays in soft agar, RUNX3-deficient A673 cells formed smaller colonies than cells expressing scrambled shRNAs. Consistent with the smaller colonies, RUNX3 suppressed cells had increased expression of the cyclin dependent kinase inhibitor p21. Taken together, these results suggest and oncogenic role for RUNX3 in Ewing sarcoma. Citation Format: Krista L. Bledsoe, Meghan E. McGee-Lawrence, Emily T. Camilleri, Andre M. Oliveira, Andre J. van Wijnen, Jennifer J. Westendorf. RUNX3 plays an oncogenic role in Ewing sarcoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1413. doi:10.1158/1538-7445.AM2014-1413

Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage‐essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt‐domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal‐derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX‐responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI‐responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma. J. Cell. Physiol. 229: 2049–2056, 2014. © 2014 Wiley Periodicals, Inc.