Abstract A48: The lncRNA HOTAIR is overexpressed in Ewing sarcoma and promotes malignant transformation through interactions with histone-modifying complexes

Abstract

Abstract Introduction: Ewing sarcoma (ES) is an aggressive primitive cancer of the bone and soft tissue in adolescents and young adults, typified by chromosomal translocations resulting in fusions of the EWS gene and an ETS family gene, most commonly FLI1. The resulting EWS-FLI1 fusion protein is the oncogenic driver in this cancer, but its expression alone does not induce malignant transformation in human mesenchymal stem cells (hMSCs), the putative cell of origin. Instead, EWS-FLI1 induces apoptosis in most recipient cell line and animal models. The lncRNA HOTAIR is upregulated in multiple types of cancer and promote tumorigenesis. However, its expression and function have not been studied in Ewing sarcoma. Methods: We used the R2 (http://r2.amc.nl) and Oncogenomics (https://pob.abcc.ncifcrf.gov/cgi-bin/JK) platforms to examine the expression of HOTAIR in Ewing sarcomas as compared to other tumors and normal tissues, as assessed in high-throughput datasets. We directly measured HOTAIR expression by RT-qPCR on total RNA from ES cell lines (n=13), primary marrow-derived hMSCs (n=2), and primary ES tumors (n=22). We repressed HOTAIR expression in cell lines by shRNA and antisense locked nucleic acid GAPmers (Exiqon), and we overexpressed HOTAIR by plasmid transfection into hMSCs. We then evaluated the phenotype of HOTAIR in these models. We performed RNA immunoprecipitation (RIP) on parental cell line extracts to identify interacting proteins with HOTAIR. We transfected mutant forms of HOTAIR into hMSCs and evaluated the effect of loss of interaction with partner proteins on the HOTAIR phenotype. We co-transfected EWS-FLI1 and HOTAIR into hMSCs to evaluate viability and phenotype. Results: HOTAIR was found to be most highly expressed in 3 independent datasets of primary Ewing sarcomas on the R2 platform as compared to all other primary tumors. In one dataset, HOTAIR was overexpressed in Ewing sarcomas (n=68) as compared to normal tissues (n=18, p<0.0001). In the Oncogenomics platform, HOTAIR was overexpressed in all ES cell lines (n=50) and primary tumors (n=72). By RT-qPCR, HOTAIR was overexpressed in 11/13 ES cell lines and 17/22 primary tumors as compared to primary hMSCs. We reduced HOTAIR expression in three ES cell lines by shRNA. We saw no effects on proliferation or chemosensitivity. However, cells with reduced HOTAIR expression had a significant reduction in anchorage-independent tumorsphere formation in soft agar, as compared to nonsilencing control cells (p≤0.01 across all three cell lines). Overexpression of HOTAIR in hMSCs this allowed anchorage independent colony formation in these cells, a phenotype not observed in the parental or vector-control cells. Treatment of the ES cell lines or HOTAIR-overexpressing hMSCs with HOTAIR-specific antisense GAPmers abrogated tumorsphere formation. We confirmed by RIP that, in ES cell lines, HOTAIR interacts with the histone-modifying proteins EZH2 at its 5' domain and LSD1 at its 3' domain, and deletion of each domain disrupts the interaction with each protein specifically. Transfection of hMSCs with HOTAIR mutants lacking either the 5' or 3' domains also failed to allow anchorage-independent tumorsphere formation. Finally, co-transfection of full-length HOTAIR with EWS-FLI1 into hMSCs allowed cell viability and tumorsphere formation, whereas expression of EWS-FLI1 alone or with mutant HOTAIR did not. Conclusions: The lncRNA HOTAIR is significantly overexpressed in Ewing sarcoma and promotes malignant transformation and tumorigenesis through mechanisms dependent on interaction with the histone-modifying proteins EZH2 and LSD1. HOTAIR may promote survival in EWS-FLI1 mediated transformation, and it may be a potential therapeutic target given its high expression in tumors and low expression in most normal tissues. Citation Format: Hasan Siddiqui, Julia Selich-Taylor, Joshua Felgenhauer, Satoru Otsuru, Edwin Horwitz, Nilay Shah. The lncRNA HOTAIR is overexpressed in Ewing sarcoma and promotes malignant transformation through interactions with histone-modifying complexes. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A48.