Abstract
BackgroundEPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.MethodsEPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.ResultsOur study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.ConclusionsTaken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.
Highlights
Erythropoietin-producing hepatocellular (EPH) receptors are clinically relevant targets in several malignancies
Upregulation of EPH-B receptors and Ephrin-B-related ligands has been found in RMS cells [18], whilst no data have yet been reported for EPH-A2- and Ephrin-A1related ligand
Western blot experiments revealed that protein expression levels and/or phosphorylation status of EPHA2, EPH-B, Ephrin-A1 (EPH-A2-related ligand) and Ephrin-B2 (EPH-B4-related ligand) were significantly increased (p < 0.01) in embryonal rhabdomyosarcoma (ERMS) cell lines in comparison to normal skeletal muscle (NSM) (Fig. 1c)
Summary
EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in childhood and adolescence, arises from undifferentiated mesenchymal cells with developing skeletal muscle features [1] In this age range, RMS includes two main histological varieties, the embryonal (ERMS) and the alveolar (ARMS) subtypes, characterized by specific genetic alterations [2,3,4]. EPH-A/Ephrin-A signalling has been shown to be essential in inducing myogenic differentiation of myoblasts through the suppression of the Ras/ERK1/2 cascade [11] Due to their physiological importance, alterations in EPH/Ephrin cascade play a central role in the pathogenesis of several diseases [12], such as cancer [13, 14], in which EPH expression levels are frequently upregulated [13,14,15], with EPH-A2 and EPH-B4 being the most widely over-expressed EPH receptors [13]. A global upregulation of EPH-B receptors and Ephrin-B ligands was previously found in RMS tumours [18], the expression levels of EPH-A receptors and Ephrin-A ligand as well as the effects of EPH/Ephrin inhibition in RMS cell biology remain unclear
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