Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The alveolar subtype (ARMS) is clinically more aggressive, and characterized by an oncogenic fusion protein PAX3-FOXO1 that drives oncogenic cellular properties. Exosomes are small, secreted vesicles that affect paracrine signaling. We show that PAX3-FOXO1 transcript alters exosome content of C2C12 myoblasts, leading to pro-tumorigenic paracrine effects in recipient cells. Microarray analysis revealed alteration in miRNA content of exosomes, affecting cellular networks involved in cell metabolism, growth signaling, and cellular invasion. Overexpression and knockdown studies showed that miR-486-5p is an effector of PAX3-FOXO1, and mediates its paracrine effects in exosomes, including promoting recipient cell migration, invasion, and colony formation. Analysis of human RMS cells showed miR-486-5p is enriched in both cells and exosomes, and to a higher extent in ARMS subtypes. Analysis of human serum samples showed that miR-486-5p is enriched in exosomes of patients with RMS, and follow-up after chemotherapy showed decrease to control values. Our findings identify a novel role of both PAX3-FOXO1 and its downstream effector miR-486-5p in exosome-mediated oncogenic paracrine effects of RMS, and suggest its possible use as a biomarker.

Rhabdomyosarcoma (RMS) is an aggressive childhood mesenchymal tumor with two major molecular and histopathologic subtypes: fusion-positive (FP)RMS, characterized by the PAX3-FOXO1 fusion protein and largely of alveolar histology, and fusion-negative (FN)RMS, the majority of which exhibit embryonal tumor histology. Metastatic disease continues to be associated with poor overall survival despite intensive treatment strategies. Studies on RMS biology have provided some insight into autocrine as well as paracrine signaling pathways that contribute to invasion and metastatic propensity. Such pathways include those driven by the PAX3-FOXO1 fusion oncoprotein in FPRMS and signaling pathways such as IGF/RAS/MEK/ERK, PI3K/AKT/mTOR, cMET, FGFR4, and PDGFR in both FP and FNRMS. In addition, specific cytoskeletal proteins, G protein coupled receptors, Hedgehog, Notch, Wnt, Hippo, and p53 pathways play a role, as do specific microRNA. Paracrine factors, including secreted proteins and RMS-derived exosomes that carry cargo of protein and miRNA, have also recently emerged as potentially important players in RMS biology. This review summarizes the known factors contributing to RMS invasion and metastasis and their implications on identifying targets for treatment and a better understanding of metastatic RMS.

Exosomes are important intercellular communication vehicles, secreted into body fluids by multiple cell types, including tumor cells. They have been demonstrated to contribute to the metastatic progression of tumor cells through paracrine signaling. Tumor exosomes contain intact and functional proteins, mRNA and miRNA that may alter the cellular environment to favor tumor growth. We evaluated the protein cargo of exosomes derived from the childhood tumor rhabdomyosarcoma (RMS) and the molecular pathways they are implicated in to decipher their role in the progression of this aggressive disease. We conducted a mass spectrometry analysis of exosome content isolated from five RMS cell lines: three of embryonal RMS (ERMS) and two of alveolar RMS (ARMS) histology and verified results by multiple reaction monitoring and western blot analyses. Results revealed 161 common proteins in ERMS-derived exosomes and 122 common proteins in ARMS-derived exosomes, of which 81 proteins were common to both subtypes. Using both PANTHER gene classification and Pathway Studio software, we assessed the perturbed biological processes and altered pathways in which the exosomal proteins are involved. The 81 commonly expressed proteins included those involved in “cell-signaling,” “cell-movement,” and “cancer.” Pathways engaging the identified proteins revealed 37 common pathways including “integrin signaling pathway,” “inflammation mediated by chemokine and cytokine signaling pathway,” and “angiogenesis.” Finally, a comparison of exosomal proteins of RMS cells with publicly available datasets from other cancer cells revealed that 36 proteins are specific and endogenous to the RMS-exosomes. Taken together, our results reveal that RMS-derived exosomes carry a protein cargo that contributes to conserved cellular signaling networks across multiple cell lines, and we also identify RMS exosome-specific proteins that should be further evaluated as possible novel biomarkers for this tumor.