Abstract A21: The PRC1 protein RING1B contributes to Ewing sarcoma tumorigenesis by blocking the NaV1.6 sodium channel and modulating the NF-κB pathway, independently of the fusion oncoprotein

Abstract

Abstract Objective: Ewing sarcoma (ES) is an aggressive and poorly differentiated tumor, commonly arising in adolescents and young adults. It is molecularly defined by a reciprocal translocation resulting in the fusion of EWS to an ETS transcription factor, being EWS-FLI the most common chimera. EWS-FLI1 up-regulates gene expression through the interaction with GGAA repeats, however, data obtained by depleting EWS-FLI1 revealed that more genes are down-regulated than up-regulated. A variety of repressive mechanisms have been described including the polycomb group (PcG) of proteins EZH2 and BMI1 that contribute to the epigenetic repressive landscape in ES. We have characterized the expression and function of the PcG RING1B in ES. Methods: 18 ES primary tissues were evaluated. The intensity of the immunostaining was evaluated and scored for the PcG RING1B, EZH2, MEL18 and BMI1. Western blot quantification was performed using Image J densitometry software. RNA was analyzed by qRT-PCR assays using SYBR Green PCR master mix from Applied Biosystems. ES cell lines (A4573, A673, SK-ES-1, TC71) were grown in standard conditions and treated with DNZep, TSA, SU5402, NSC87877, S3I-201 and BMS-345541 for 24 hours. Mock, RING1B, BMI1, FLI1 and EZH2 shRNA and siRNAs were generated using standard methods. Microarray analysis was performed according to protocols from Ambion and Affymetrix, and hybridized to GeneChip Human Gene 2.0 ST Array (Affymetrix). Each experiment was performed in triplicate at least three times. Statistical analysis was performed using the Student's t-test with a p < 0.05 as significant. Results: RING1B was highly and universally expressed in all primary ES tumor specimens. In ES cell lines, RING1B mRNA levels were not affected by oncogene down-regulation or treatment with differentiation agents (ATRA; BHA). When RING1B was depleted in ES cell lines by retroviral shRNA, heme biosynthesis and hematopoiesis were 2 of the canonical pathways identified and the set of genes regulated by RING1B unique. Among the most up-regulated genes upon RING1B depletion stand FGF14 and SCN8A. Repression of the 2 components of the NaV1.6 channel was proved specific for RING1B and functionally relevant in ES. Upon RING1B depletion, NF-κB was the most significantly altered pathway identified. Expression of the NF-κB protein p105 (and p50) was found enhanced and RING1B depletion sensitized specifically ES cells to apoptosis by FGFR-SHP2 and STAT3 blockade. Conclusion: we show that RING1B is pivotal in ES cells, independent of the fusion oncogene, likely reflecting traits of the cell of origin. Citation Format: Inmaculada Hernandez-Muñoz, Elisabeth Figuerola, Sara Sanchez-Molina, Jaume Mora. The PRC1 protein RING1B contributes to Ewing sarcoma tumorigenesis by blocking the NaV1.6 sodium channel and modulating the NF-κB pathway, independently of the fusion oncoprotein. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A21.