Abstract 2066: The functional noncoding genome in childhood sarcomas plays important regulatory roles in tumor pathogenesis
Authors: Mitra Sheetal A. , Mitra Anirban P. , Liu Yang , Buckley Jonathan D. , Triche Timothy J. 
Published: August 2018

Pediatric soft tissue sarcomas such as rhabdomyosarcomas (RMS) and Ewing sarcomas (ES) occur in children and adolescents. These tumors typically have a very primitive stem cell-like appearance with few mutations, thus underscoring the importance of other genetic and epigenetic mechanisms responsible for initiating and maintaining tumorigenesis. Noncoding RNAs (ncRNA) are known to play an important role in development and cancer and hence we hypothesized that they would regulate the genomes of these developmental sarcomas. Whole-transcriptome profiling was performed on primary tumors from 200 ES, and 80 RMS patients. Cox regression models were used to generate prognostic metafeatures (MF) for the RMS cohort. A 30-protein-coding MF (pcMF, p= 0.001) and a 34-noncoding MF (ncMF, p&lt;0.001) both predicted survival, but the accuracy of the ncMF was higher than the pcMF (96% vs. 71%, p&lt;0.001). Pathway analyses showed association of pcMF with musculoskeletal development and signaling pathways while ncMF enriched for cellular assembly, cell cycle, apoptosis, and cancer-associated functions. Further, the ncRNA profile was driven by expression of a 600kb very long noncoding RNA (vlncRNA), expressed at high levels only in surviving patients. For ES, we identified two highly expressed tumor-specific ncRNAs, a long ncRNA (lncRNA, 2.6kb) FEZF1-AS1, and another, 400kb vlncRNA, C9orf92. While both the ncRNAs were regulated by EWS-FLI1, only FEZF1-AS1 was directly regulated by the fusion protein binding to GGAA repeats in its promoter. In vitro Matrigel assays and tail vein mouse models of tumor metastasis showed that FEZF1-AS1 promoted tumor invasion while the C9orf22 vlncRNA inhibited tumor growth and metastasis. FEZF1-AS1 is present in the human fetal brain and a subset of genes induced by both EWS-FLI1 and FEZF1-AS1 allow for cell organization and neuronal development. Chromatin immunoprecipitation assays show that FEZF1-AS1 modifies histone activating marks across the ES genome while RNA-IP assays indicate binding of the lncRNA to chromatin modifying proteins such as EZH2 and HDACs. RNA-ISH and protein IF showed co-localization of vlncRNA with AP1. vlncRNA overexpression decreased protein levels of the MET oncogene in ES metastatic cell line. Matrigel assays showed that ES cells with decreased MET expression had reduced invasiveness, a feature associated with metastasis and poor outcome. Outcome data for 140 ES patients showed high expression of the 400kb vlncRNA correlated with increased survival rates (p&lt;0.01). In conclusion, our studies demonstrate that noncoding RNAs of diverse type are highly functional in childhood sarcomas and predict outcome better than any coding RNA. Functional studies indicate that they regulate necessary pathways of cell growth and metastasis in ES pathogenesis. These findings highlight the importance of the regulatory noncoding genome in childhood tumors.

Citation Format: Sheetal A. Mitra, Anirban P. Mitra, Yang Liu, Jonathan D. Buckley, Timothy J. Triche. The functional noncoding genome in childhood sarcomas plays important regulatory roles in tumor pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2066.