Abstract 2501: Identification and characterization of key players in the Ewing Sarcoma metastatic process

Abstract

Abstract Metastasis at diagnosis is the most adverse prognostic factor in Ewing Sarcoma (ES), drastically decreasing patients’ overall survival down to <30%1. However, mechanisms leading to metastasis in this disease remain poorly understood. To deepen our knowledge on the main players in ES metastasis, Microarray (Clariom™ D Array, Affymetrix) and phosphoarray (Phospho Explorer Antibody Arrays, Full Moon Biosystems) analyses were performed on primary tumours (n=5) and metastases (n=12) samples from an spontaneous metastatic ES mouse model2. After normalisation and bioinformatics analysis, a list of differentially expressed genes/proteins between the 2 sample groups was obtained. Candidates were validated by qPCR (TaqMan™ probes) and Western Blot, and further characterised in vitro through loss-of-function phenotypic assays (proliferation, clonogenic and migration) using siRNAs and/or inhibitors. Several genes and proteins of interest, including LOXHD1, confirmed to be overexpressed in metastases samples compared to primary tumours as identified by microarray/phosphoarray analyses and qPCR/Western Blot validation. Also, candidates subjected to transient loss-of-function phenotypic assays significantly decreased in all cases tumorigenic and/or metastatic capabilities of ES cells when suppressed. Additionally, LOXHD1 showed upregulation in sphere-growing ES cells, validating its role in ES hypoxia and metastasis as demonstrated previously3. Using stable knock-out models of the genes of interest, the exact mechanism and pathways in which these candidates play a role in ES metastasis will be further studied in vitro and in vivo. Also, their clinical relevance will be explored in several cohorts of patients by immunohistochemistry on Tissue Microarrays. [1] Grünewald, TGP et al. 2018, Nat Rev Dis Prim, 4(1), pp.1-22. [2] Lagares-Tena, L et al. 2016, Oncotarget, 7(35), pp.56889-903. [3] Deng, Q et al. 2021, bioRxiv 2021.02.22.432287. Citation Format: Sara Sanchez Serra, Mariona Chicon Bosch, Judith Besalú Velazquez, Susana Maqueda Marcos, Paola Monaco, Laura Santana Viera, Roser Lopez Alemany, Oscar M. Tirado. Identification and characterization of key players in the Ewing Sarcoma metastatic process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2501.