The PAX3-FOXO1 oncogene alters exosome miRNA content and leads to paracrine effects mediated by exosomal miR-486

Abstract

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.

Signaling pathways in Rhabdomyosarcoma invasion and metastasis

“…RMS cells also express chemokine receptors such as CXCR4 and leukemia inhibitory factor receptor (LIFR) which respond to stimulation by SDF-1 and LIF respectively allowing their homing into distant organs. Sphingosine-1-phosphate (S1P) released by RMS cells subsequently enhances RMS invasion and metastasis through autocrine stimulation of RMS cells and paracrine stimulation of stromal cells One of the miRNAs differentially expressed in exosomes, miR-486-5p, is a downstream target of the PAX3-FOXO1 fusion oncoprotein and indeed exhibits higher levels of expression in FPRMS-derived exosomes [132,133]. Ectopic expression of the fusion oncoprotein PAX3-FOXO1 in C2C12 mouse myoblasts results in an increase in miR-486-5p expression level in both cells and exosomes, and treatment of recipient fibroblasts with FPRMS-derived exosomes increases cell migration and invasion in a miR-486-5p expression-dependent manner [133].…”

“…Efforts should also be made to detect biomarkers that are predictive of RMS metastasis and response to treatment; this would allow a more accurate prognosis of disease. One such promising biomarker may be miR-486-5p, where in a limited number of samples, its levels seemed to be higher in exosomes derived from FPRMS patient serum, deserving further validation in larger sample sizes [133]. Additionally, proteins that are commonly expressed within FPRMS-and FNRMSderived exosomes and have been implicated in cancer metastasis, such as CD147, are interesting candidates for further evaluation as possible therapeutic targets [145][146][147].…”

Effects of the Oncoprotein PAX3-FOXO1 on Modulation of Exosomes Function and Protein Content: Implications on Oxidative Stress Protection and Enhanced Plasticity

“…HUVEC (human umbilical vein endothelial cells) and HEK293T (human embryonic kidney) cells were also purchased from ATCC. As described previously, viral supernatants were produced by transfecting 293T cells with MSCV-P3F or MSCV-GFP vectors using calcium phosphate ( 18 ). C2C12 cells were then transduced in suspension with either MSCV-GFP viruses (forming Ctrl-C2C12 cells) or MSCV-P3F (forming P3F-C2C12 cells) at 32°C, 1250 × g for 1 h with 8 μg/ml Polybrene (hexadimethrine bromide; Sigma), and sorted using FACS Aria SORP cell sorter (BD, New Jersey, United States) after selection with 2 μg/ml Puromycin (Abcam).…”

“…Briefly, Ctrl-C2C12 cells, P3F-C2C12 cells, JR1 and Rh30 cells were incubated in exosome-free medium prepared by ultracentrifugation at 100,000 × g , overnight at 4°C. Exosomes were then isolated from the conditioned media by differential ultracentrifugations ( 18 ): 300 × g for 10-min 2,000 × g for 20-min centrifugation, and then 10,000 × g for 30 min. Finally, ExoQuick Exosome Precipitation Solution (System Biosciences, mountainite, CA, United States) was added to the resulting supernatant, and stored overnight at 4°C to allow exosome precipitation.…”

Preparing the Bone Tissue Regeneration Ground by Exosomes: From Diagnosis to Therapy

“…These signaling molecules include proteins, lipids, and noncoding RNAs, and their configuration is highly dependent on the status of the releasing cell and its microenvironment. Therefore, any changes within the microenvironment of a given tissue will be reflected in the content of its released exosomes [ 29 , 30 , 31 ]. In a number of pathologies, patient blood or body fluid samples were found to contain disease- specific micro RNAs (miRNAs), including bone-disease specific miRNAs [ 32 , 33 ].…”