Ewing sarcoma (ES) is a universally metastatic tumor of bone and soft tissue. Five-year survival in patients with metastases at diagnosis is about 20% despite intense treatment strategies. 85% of these tumors harbor a chromosomal translocation resulting in a fusion gene, EWS-FLI1. EWS-FLI1 imparts an oncogenic phenotype by modifying critical genes in signaling pathways, thus allowing for uninhibited cell growth, invasion, and metastasis while evading differentiation. EWS-FLI1 is thus an ideal therapeutic candidate, but targeting this fusion protein has proven difficult. In an attempt to better understand ES biology and identify candidate therapeutic targets downstream of EWS-FLI1, we profiled RNA transcription in over 140 ES cases and compared the data to other pediatric cancers. Among the most highly expressed was a recently discovered long non-coding RNA (lncRNA), FEZF1-AS1, in addition to protein-coding genes such as NR0B1, and CD99, which are known to be important targets of the EWS-FLI1 chimeric protein. FEZF1-AS1 expression was specific for ES and was increased more than a hundred fold in ES when compared to other pediatric tumors. Chromatin immunoprecipitation (ChIP) studies along with luciferase promoter assays showed that EWS-FLI1 induces FEZF1-AS1 expression by binding to promoter GGAA repeats. shRNA-mediated lncRNA knockdown did not affect cell growth or survival. Instead, metastatic potential was markedly reduced, based on Matrigel assays. Conversely, ES cells transfected to overexpress FEZF1-AS1 engrafted better in mice and metastasized faster than control cells. Expression profiling of lncRNA-knockdown cells showed that the lncRNA upregulated genes were involved in cell movement and cell organization. ChIP-seq documented significant reduction in H3K4me3 and the H3K27ac histone marks in the promoters of genes that were downregulated as a result of lncRNA-knockdown. In order to document FEZF1-AS1 function, we delivered FEZF1-AS1-specific antisense oligonucleotide (ASO) and GFP-ASO in anti-CD99-targeted-nanoparticles to ES cells. An efficient uptake of nanoparticles and lncRNA-ASO delivery to ES cells led to a 70% decrease in lncRNA expression in cells within four hours of initiating treatment. LncRNA-ASO-targeted ES cells maintained low lncRNA expression for 72 hours and showed decrease invasion on matrigel assays. The cells tolerated the treatment well with no adverse effect on cell survival and proliferation. These results suggest that FEZF1-AS1 is a highly functional and ES-specific lncRNA that can be an important therapeutic target, reduced expression of which markedly decreases metastasis of ES tumors. The primary mechanism of action appears to be histone acetylation that activates genes involved in cell migration. The nanoparticle data indicates that FEZF1-AS1 is also a potential therapeutic target, via targeted delivery of inhibitory ASOs.
Citation Format: Sheetal A. Mitra, Anirban P. Mitra, Jon O. Nagy, Jonathan D. Buckley, Timothy J. Triche. Metastasis promoting long non-coding RNA, FEZF1-AS1, is an important therapeutic target for Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4905. doi:10.1158/1538-7445.AM2017-4905