Abstract 3672: Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3-mediated regulation of CD164-CXCR4 axis impacts aggressiveness of Ewing sarcoma

Abstract

Abstract INTRODUCTION: The genomic stability of primary and metastatic Ewing sarcoma (EWS), the second most common primary bone tumor in pediatric age, evokes epigenetic or mRNA translational reprogramming as mechanisms involved in tumor progression. The oncofetal RNA-binding protein (RBP) insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 (IGF2BP3) controls stability and translation of transcript targets and represents a major determinant of EWS prognosis and cell migration. Reported IGF2BP3 mRNA targets involved in cancer cell motility include CD164 (endolyn), a cell surface receptor for sialomucin which induces migration interacting with chemokine receptor CXCR4. Here we aim to dissect the role of IGF2BP3 as a critical regulator of CD164-CXCR4 axis in relation to EWS malignancy. MATERIAL AND METHODS: Ribo-immunoprecipitation (RIP) assay and qRT-PCR were employed to test IGF2BP3-mRNAs interactions. CD164 and CXCR4 expression was assessed by qRT-PCR and western blot in stable IGF2BP3 knockdown EWS cellular models compared to controls. Soft-agar growth, migration and CXCR4 expression were investigated after treatment with anti-CD164 siRNA or scramble control. Cell growth and migration in hypoxic compared to normal conditions was assessed by Trypan Bleu and transwell migration assays after exposure of cells with differential expression of IGF2BP3 to hypoxia mimetic agent CoCl2 (100 µM). Samples of 79 paraffin-embedded EWS primary tumors were included in a tissue microarray (TMA) and processed for immunohistochemical evaluation of IGF2BP3, CD164 and CXCR4 to establish correlations (Chi-square test). RESULTS: Within the panel of reported IGF2BP3 transcript targets involved in cancer invasiveness (CD44, MMP9, CD164), CD164 was the most abundantly enriched transcript after immunoprecipitation with anti-IGF2BP3 antibody in EWS cells. IGF2BP3 knockdown, beyond decreasing the invasive potential of EWS cells, down-regulated CD164 and, interestingly, its partner CXCR4, a major mediator of stress-response including hypoxia. Functional studies confirmed that CD164 silencing impaired soft-agar growth and migration of EWS cells and decreased the expression of CXCR4. Accordingly, presence of IGF2BP3/CD164/CXCR4 axis favored cell growth and migration in hypoxic conditions. In EWS patients, a direct correlation between IGF2BP3 and CD164 (p-value minor than 0.001) or CXCR4 (p-value equal to 0.035) was found, confirming the soundness of the in vitro results at clinical setting. CONCLUSIONS: This study describes CD164 as a novel mediator of EWS aggressiveness and it highlights a connection between IGF2BP3 and the CD164/CXCR4 axis that may further explain the oncogenic role of IGF2BP3 in this tumor. (Grants: AIRC IG2016_18451 and RF Ministry of Health PRWEB 7153 to KS) Citation Format: Caterina Mancarella, Maria Cristina Manara, Arthur M. Mercurio, Katia Scotlandi. Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3-mediated regulation of CD164-CXCR4 axis impacts aggressiveness of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3672.