Abstract 3201: Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 predicts poor prognosis and promotes cell proliferation in Ewing sarcoma

Abstract

Abstract INTRODUCTION: Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 (IGF2BP3, also known as IMP3) is an oncofetal protein which binds RNA thus influencing the transcript target fate. IGF2BP3 is de novo synthesized in malignancies where it promotes proliferation, drug resistance and metastases. Moreover, IGF2BP3 represents a promising indicator of outcome in several cancers. Ewing sarcoma (EWS) is a rare bone and soft tissue tumor where the IGF system plays a pivotal role. This study aimed to investigate the relevance of IGF2BP3 in EWS and verify its value as a prognostic biomarker. MATERIAL AND METHODS: Total RNA was extracted from two independent cohorts of 30 and 109 snap-frozen tissues from localized primary EWS samples with more than 5 years follow-up. Microarray analysis (Affymetrix) was performed in the cohort of 30 patients while IGF2BP3 expression was analysed by qRT-PCR (Applied Biosystem) in the cohort of 109 cases. Prognostic value of IGF2BP3 was evaluated both in univariate and multivariate analysis. IGF2BP3 expression was assessed by qRT-PCR and western blot in a panel of 14 EWS cell lines. Plasmids for IGF2BP3 silencing or over-expression were transfected in A673 or LAP-35 and IOR/CAR EWS cells, respectively. Soft-agar growth of cell lines with differential IGF2BP3 expression was evaluated. catRAPID express human web server was employed for prediction of novel IGF2BP3/mRNAs interactions. IGF-2 and ABCF1 expression levels were analysed in vitro and in clinical specimens. RESULTS: Microarray results evidenced that IGF2BP3 higher expression correlated with a worse outcome (p-value 0.0002). This data was confirmed in the cohort analysed by qRT-PCR both considering univariate (p-value 0.001) and multivariate analysis (HR:2.83. IC95% [1.22-6.53].p = 0.015). In vitro, higher IGF2BP3 expression correlated with an increased capability of growth in anchorage-independent conditions and IGF2BP3 knockdown significantly decreased soft-agar growth of EWS cells. Oncogenic potential of IGF2BP3 was not found to be mediated through IGF-2-dependent mechanism but ABCF1, an ABC family member with a key role in translation initiation, was predicted as a target of IGF2BP3 by in silico analysis. Accordingly, IGF2BP3 loss- or gain-of-functions approaches induced down- or up-regulation of ABCF1 protein levels, respectively. In addition, a direct correlation between IGF2BP3 and ABCF1 was found in 109 EWS patients (p-value minor than 0.0001). Functional studies on the role of ABCF1 in EWS will follow. CONCLUSIONS: IGF2BP3 represents an indicator of prognosis in EWS therefore its detection may be of help to stratify patients according to risk. We also provided evidences for IGF2BP3 in the regulation of EWS aggressiveness and identified ABCF1 as a new putative mediator of IGF2BP3 malignant effects. (Grants: FIRB RBAP11884M_005 and AIRC IG2013_14049 to KS) Citation Format: Caterina Mancarella, Linda Calzolari, Stefano Ferrari, Davide Donati, Piero Picci, Katia Scotlandi. Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 predicts poor prognosis and promotes cell proliferation in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3201.